Journal of Ethnopharmacology 119 (2008) 686–699 Contents lists available at ScienceDirect Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jethpharm The biological activity and chemistry of the southern African Combretaceae J.N. Eloff ∗ , D.R. Katerere 1 , L.J. McGaw Phytomedicine Programme, Department of Paraclinical Sciences, Faculty of Veterinary Science, University of Pretoria, Private Bag X04, Onderstepoort 0110, South Africa a r t i c l e i n f o Article history: Received 16 May 2008 Received in revised form 25 July 2008 Accepted 30 July 2008 Available online 30 August 2008 Keywords: Antibacterial Antifungal Combretaceae Combretum South Africa Terminalia a b s t r a c t Aim of the study: Members of the Combretaceae family are widely traded in the traditional medicine market in southern Africa. The family is also used for medicinal purposes in the rest of Africa and Asia for close to 90 medicinal indications. Many of these indications are related to treating infections. This contribution summarizes work done to date and identifies avenues for future research. Materials and methods: Substantial work has already been done on the chemistry of especially Combretum and Terminalia species over many years. During the last decade we have focussed on bio-assay guided isolation of biologically active compounds with the aim of producing new effective antimicrobial products. Results and discussion: Methods developed to facilitate this process and data on 25 compounds isolated from 7 species are presented. The large majority of compounds isolated were known, but the biological activities were not known. In practically all cases the antibacterial or antifungal activity of compounds isolated were much lower than expected from the activity of the crude extracts. It appears that synergism plays a role in antimicrobial activity of plant extracts and that the hope of isolating a single compound that can be used as a new agent to address antibiotic resistance has been frustrated. By simple manipulation such as selective extraction the activity of some crude extracts could however, be increased substantially and this offers a new approach to address antibiotic resistance via the herbal medicine industry. Practically all extracts obtained using intermediate polarity extractants had reasonable to very good activity with MICs as low as 40 ␮g/ml, validating the traditional use for infectious diseases. Aqueous extracts however, generally had hardly any activity. Conclusions: The Combretaceae contains a diversity of antimicrobial compounds. Because poor people usually have only water available as extractant, it raises the question how plants growing in poor rural communities can be used to treat infections more effectively, and what the mechanism of activity of aqueous extracts used to treat infections in traditional medicine are. © 2008 Elsevier Ireland Ltd. All rights reserved. 1. Introduction 1.1. Occurrence and taxonomy Members of the Combretaceae family occur widely in tropical and subtropical areas. The family is placed in the Myrtales and consists of 20 genera and c. 300 spp. (Tan et al., 2002). The largest genera are Combretum, Terminalia and Quisqualis. The other genera are Anogeissus, Buchenavia, Bucida, Calopyxis, Calycopteris, Conocarpus, Abbreviations: C, Combretum; DPPH, diphenylpicrylhydrazyl; INT, tetrazolium violet; LD50 , dose that was lethal to 50% of organisms; MIC, minimal inhibitory concentration; MS, mass spectroscopy; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide; NMR, nuclear magnetic resonance; Pt, pteleopsis; T, Terminalia; TLC, thin layer chromatography. ∗ Corresponding author. Tel.: +27 12 5298244; fax: +27 12 5298525. E-mail address: kobus.eloff@up.ac.za (J.N. Eloff). 1 Current address: PROMEC Unit, Medical Research Council, P.O. Box 19070, Tygerberg 7505, South Africa. 0378-8741/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2008.07.051 Dansiea, Guiera, Laguncularia, Lumnitzera, Macropteranthes, Melostemon, Pteleopsis, Quisqualis, Strephonema, Terminaliopsis and Thiloa. From the first checklist of flowering plants in sub-Saharan Africa (Klopper et al., 2006), it is clear that in Africa the family is mainly tropical. Ignoring subspecies about 133 Combretum species occur in tropical Africa (North of the tropic of Capricorn) and 31 species occur only or also in subtropical Africa. The ratio for Terminalia species is similar (32 and 7, respectively). The Combretum spp. occurring in southern Africa is presented in Table 1 (Carr, 1988). Carr (1988) also listed the following Terminalia species: Section Abbreviatae—T. prunioides, T. randii, T. stuhlmannii; Section Psidioides—T. brachystemma, T. sericea, T. trichopoda; Section Platycarpae—T. gazensis, T. phanerophlebia, T. mollis, T. sambesiaca, T. stenostachya. The taxonomic treatment used here is that of Klopper et al. (2006). The growth form varies from forest trees more than 50 m high to shrubs and lianas. The large trees and lianas occur in forests while shrubs occur in grassland and mangroves. The wide variation in flowers, fruits and vegetative shoot morphology complicates J.N. Eloff et al. / Journal of Ethnopharmacology 119 (2008) 686–699 Table 1 Taxonomic relationship of Combretum spp. occurring in southern Africa (Carr, 1988) Subgenus Combretum Loefl Section Hypocrateropsis: C. celastroides [4/5], C. imberbe [10], C. padoides [18] Section Combretastrum: C. umbricola Section Angustimarginata: C. caffrum [3], C. erythrophyllum [8], C. kraussi [11], C. nelsonii [17], C. vendae, C. woodii [21] Section Macrostigmate: C. engleri, C. kirkii, C. mkuzense [13] Section Metallicum: C. collinum [6] Section Glabripetala: C. fragrans Section Spathulipetal: C. zeyheri [22] Section Ciliatipetala: C. albopunctatum, C. apiculatum [1], C. edwardsii [7], C. moggii [14], C. molle [15], C. petrophilum [20], C. psidioxides Section Fusca: C. coriifolium Section Breviramea: C. hereroense [9] Section Elaeagnoida: C. elaeagnoides Subgenus Cacoucia Section Lasiopetal:a C. obovatum Section Conniventia: C. microphyllum [12], C. paniculatum [19], C. platypetalum Section Oxystachya: C. oxystachytum Section Poivrea: C. bracteosum [2], C. mossambicense Section Megalantherum: C. wattii Numbers refer to extracts analyzed in Fig. 1. identification of species (Stace, 1969). Carr (1988) described the morphology, taxonomy and propagation of southern African members of the Combretaceae. This included species occurring in Zimbabwe as part of tropical Africa (Table 1). 1.2. Ethnomedical use and commercial value of Combretaceae Many species of the Combretaceae are used medicinally in several continents in the world. Traditional healers in eastern and southern Africa have used Combretum and Terminalia species, for many applications including treating abdominal disorders, backache, bacterial infections, bilharzia, cancer, chest coughs, cleansing the urinary system, colds, conjunctivitis, constipation, diarrhoea, dysentery, dysmenorrhoea, earache, fever, gastric ulcers, general weakness, gonorrhoea, headaches, heart diseases, hookworm, hypertension, jaundice, leprosy, nosebleeds, oedema, pneumonia, skin diseases, sore throats, stomach and gastric problems, swelling caused by mumps, syphilis, toothache, venereal diseases (OliverBever, 1986; Iwu, 1993; Hutchings et al., 1996; Neuwinger, 1996; Fyhrquist et al., 2002). In West Africa Burkhill (1985) listed many more traditional medicinal uses of the Combretaceae. This includes acute enteritis, anthelminthic, anuria, aphrodisiac, ascitis, beriberi, blennorrhoea, burns, catarrh, chew sticks, cholagogue, cicitrisant, cilic, colitis, constipation, craw-craw, dental caries, diuretic, dropsy, enteralgia, expectorant, eye diseases, general fatigue, gingivitis, guinea-worm, haematuria, haemorrhoids, helminthiasis, hiccups, insanity, internal parasites, jaundice, loss of appetite, low backache, lumbago, malaria, menorrhagia, nausea, poison antidote, post circumcision haemorrhage, purgative, ringworm, sarcoma tumours, scorpion sting relief, sore throat, temporary insanity, thrush, tooth ache, tuberculosis, tumours, vermifugal, wasting, yaws on foot soles, yellow fever. There can be no doubt that the Combretaceae forms an important part of Africa’s traditional medicine. The West African Combretum micranthum was so widely used as a general panaceae and known for its diuretic, febrifugal and digestive properties that the common name for the species kinkeliba has become a word synonymous with medicine in some languages. It is interesting that in Ivory Coast and Nigeria Combretum molle, occurring widely in southern Africa, has been used in the absence of Combretum micranthum. The medicinal importance of Terminalia and Combretum species was confirmed when Terminalia sericea and 687 Combretum micranthum were selected among the 50 most important African medicinal plant species by the Association for African Medicinal Plant Standards (http://www.aamps.org). Combretum micranthum was exported to Europe after it value was discovered by expatriates living in West Africa. Apparently Terminalia sericea bark was also exported to Europe from Mozambique (Rukangira 2000, as quoted by Fyhrquist, 2007). In Durban, South Africa Combretaceae was one of the main plant families used in KwaZulu-Natal in traditional medicine with an average of 20.2 metric tons traded per year (Cunningham, 1990). Several of the uses of Combretaceae point towards possible anti-infective activity, including antibacterial, antifungal and antiparasitic activity. A search on Google Scholar for “Combretum and traditional” gave 1910 hits and for “Terminalia and traditional” gave 4920 hits. A large number of the latter may refer to Terminalia catappa a widely used in Asian species, but the numbers indicate the wide traditional use of these two genera. 1.3. Motivation for studying Combretaceae The Phytomedicine Programme at the University of Pretoria has been investigating species of the Combretaceae for several years. The motivation for the selection of the plant family for research is described elsewhere (Eloff, 1998c). In addition to the extensive ethnobotanical use of the family in Africa listed above, the pharmaceutical company Noristan found evidence of antibacterial activity in Combretum erythrophyllum extracts (Dr. B. Fourie, personal communication). Earlier work done on Combretum and Terminalia species was mainly on the chemistry. The overall aim of our research was to evaluate species in this important family for anti-infective activities with the motivation of discovering compounds that could be used in the pharmaceutical industry. Additional aims were to validate the ethnomedicinal use of Combretaceae spp. and to investigate the possibility that extracts could be used in the primary health care of rural people. The current knowledge on the chemistry of the family will be discussed followed by a discussion of compounds isolated by bioassay-guided fractionation and the biological activity of the isolated compounds and extracts. For the application of plant extracts to treat human or animal infections not only the antimicrobial activity of isolated compounds or extracts, but also the toxicity of these extracts and animal studies are required and this will be addressed. 2. Chemistry of the Combretaceae The Combretaceae is the source of a wide range of tannins, flavonoids, terpenoids and stilbenoids. The latter have generated immense interest in this family because of their chemical simplicity, which belies their therapeutic potential. The major compounds isolated from Combretaceae are discussed in greater detail in Sections 2.1–2.3. 2.1. Stilbenoids and phenanthrenes These compounds were first isolated in Zimbabwe in the 1970s from C. apiculatum, C. psidioides and C. molle (Letcher and Nhamo, 1971, 1972, 1973; Letcher et al., 1972). A decade later Pettit and co-workers isolated similar compounds from the South African C. caffrum and discovered potent anti-tubulin activity and action against murine P388 leukaemia cells in vitro (Pettit et al., 1982, 1987, 1988a,b). Stilbenoids from the Combretaceae have been named combretastatins and are designated A, B, C and D. Combretastatin A 688 J.N. Eloff et al. / Journal of Ethnopharmacology 119 (2008) 686–699 Table 2 Examples of structural classes isolated from members of the Combretaceae Stilbenoids and phenanthrenes Primary structure of Combretastin A isolated from C. caffrum, C. kraussi (Pettit et al., 1982; Rogers and Verotta, 1996) 9,10-Dihydrophenanthrenes isolated from C. apiculatum, C. molle, C. caffrum (Pettit et al., 1982; Rogers and Verotta, 1996; Letcher and Nhamo, 1971, 1972) Polyphenols Arjunolone from T. arjuna (Kumar and Prabhakar, 1987) Punicalagin from T. oblongata (Doig et al., 1990) Cyclobutane dimmer from C. apiculatum and C. albopunctatum (Katerere et al., 2004) Terpenoids Arjunolic acid from C. molle and T. arjuna (Panzini et al., 1993; Kumar and Prabhakar, 1987) J.N. Eloff et al. / Journal of Ethnopharmacology 119 (2008) 686–699 689 Table 2 (Continued ) Mollic acid and derivatives from C. molle (Panzini et al., 1993) Cycloartane dienone lactone from C. erythrophyllum (Rogers and Verotta, 1996) Imberbic acid dirhamnoside compound isolated from T. stulhmanii (Katerere et al., 2003) Alkaloids 4-Hydroxyproline betaine, an alkaloid from the leaves of C. micranthum (Ogan, 1972) which possesses an ethylene bridge between the two benzyl rings (i.e. stilbene) (Table 2) and combretastatin B with an ethane bridge (dihydrostilbene) are the most common. These compounds have since been isolated from C. kraussi, C. molle, C. psidioides, C. apiculatum, C. collinum and leaves of C. woodii (Malan and Swinny, 1993; Schwikkard et al., 2000; Katerere, 2001; Eloff et al., 2005b) and also from the leaves of Cannabis sativa (Crombie and Crombie, 1975) and some Orchidaceae species (Juneja et al., 1987; Majumder et al., 1999). The phenanthrenes are phenolic compounds with three fused rings and have been isolated from many families viz. Orchidaceae, Horaceae and Combretaceae (Miyase and Ueno, 1991) (Table 2). They are either precursors or products of the stilbene metabolic pathway and have been isolated from several Combretum taxa including C. hereroense, C. molle, C. apiculatum and C. collinum (Letcher and Nhamo, 1971, 1972, 1973; Letcher et al., 1972; Pettit et al., 1988b; Malan and Swinny, 1993; Katerere, 2001). The dihydrophenanthrenes and phenanthrenes from C. caffrum showed good activity against murine P388 lymphocytic leukaemia cell lines (Pettit et al., 1988b). Phenanthrenes and stilbenes are well recognized as phytoalexins (Hart, 1981; Kovács et al., 2008). This may be one explanation of the antimicrobial activity of the Combretaceae described elsewhere (Eloff et al., 2005a). Structure–activity relationships appear to be important in the activity of stilbenes and phenanthrenes because substituents determine the planarity of the molecules, which is essential for drug–receptor interactions (Rogers and Verotta, 1996; Katerere, 2001). 2.2. Polyphenols Various types of flavonoids have been reported from Terminalia arjuna (Kumar and Prabhakar, 1987), C. leprosum (Facundo et al., 1993) and C. erythrophyllum (Martini et al., 2004) amongst others. Chalcones were isolated from the leaves of C. apiculatum as well as from an extract of the leaves and fruit of C. albopunctatum (Katerere et al., 2004; Serage, 2004). Of interest was the isolation of two ␣-truxillic chalcone dimers from the latter. The dimers were not active in antimicrobial assays when compared to their constituent monomeric units. Montaudo et al. (1974) and Toda et al. (1998) have previously reported that these compounds are photodimers, while Seidel (1999) isolated one such dimer from Annonaceae. Flavanones and flavones have also been isolated and appear to be ubiquitous constituents (as would be expected) in the leaf extracts of Combretum species. Of interest was the isolation of pinocembrin from C. apiculatum (Katerere, 2001; Serage, 2004). This compound is known to be responsible for the antimicrobial activity of propolis (Castaldo and Capasso, 2002). A number of elaborate tannins (e.g. diphenoyl-gallagylglucose and ellagitannin and their derivatives) have been isolated mainly from Asian Terminalia species, in particular T. oblongata, T. clamansanai, T. catappa and T. cheluba (Tanaka et al., 1986, 1991; Liu et al., 1996). While in the past tannins were viewed as possessing non-selective biological activity, primarily ability to precipitate proteins, recent evidence suggests that the bioactivity of tannins varies with their structural differences and ranges from antimutagenicity, inhibition of tumour promotion, anti-HIV, antimicrobial, 690 J.N. Eloff et al. / Journal of Ethnopharmacology 119 (2008) 686–699 antileishmanial to antioxidant activity (Okuda et al., 1989; Muetzel and Becker, 2006; Kolodziej and Kiderlen, 2005). Flavans have been reported only from Terminalia bellerica and T. argentea (Garcez et al., 2003). However since they are precursors of condensed tannins, they probably occur more widely in Combretaceae. Anolignan B was isolated from Terminalia sericea and Anogeissus acuminanta (Eldeen et al., 2006). This compound showed activity against both Gram-negative and Gram-positive bacteria and HIV-1 reverse transcriptase. 2.3. Terpenoids The Combretaceae has yielded mainly pentacyclic triterpenoids varying from oleanoic and ursanoic acids to friedelins, cycloartanes and dammaranes. Arjunolic acid and glycosides have been isolated from C. molle and T. arjuna (Kumar and Prabhakar, 1987; Panzini et al., 1993). Sericic acid and sericoside have been found from the roots of T. sericea (Eldeen et al., 2006). Friedelin, epifriedelin and betulinic acid from the bark of C. imberbe and an oleanene-based pentacyclic triterpene (imberbic acid) and its glycosides have been reported (Rogers, 1988, 1989; Rogers and Subramony, 1988; Angeh et al., 2007a). Other oleanene-type pentacylic triterpenoids bearing 29-carboxy and 1␣-hydroxy substituents have been isolated from C. molle, C. edwardsii, C. eleagnoides, C. apiculatum, C. kraussi, C. padoides and Anogeissus leiocarpus (Rogers and Verotta, 1996; Katerere et al., 2003; Angeh et al., 2007b; Chaabi et al., 2008). These compounds demonstrate the close chemotaxonomic relationships among the species and also between African and South American Combretum species (Facundo et al., 1993; Rogers, 1995). Cycloartane-type triterpenoids have been isolated from C. erythrophyllum (Rogers and Verotta, 1996) and C. quadrangulare (Banskota et al., 2000) while acidic dammarane arabinofuranosides have been reported from C. rotundifolium (Facundo et al., 1993). Co-occurrence of tetracyclic and pentacylic classes of these triterpenoids is unusual but C. molle contains both (Panzini et al., 1993). Katerere et al. (2003) reported on the isolation of acetylated rhamnosides of 1,3-hydroxylated pentacyclic triterpenoids from C. imberbe and T. stulhmanii. One compound in particular was isolated simultaneously from both plant species, thus cementing their close evolutionary relationships. These compounds have good activity against Mycobacterium fortuitum, which is being further investigated. 2.4. Alkaloids and nitrogen containing compounds Until recently, reports of the presence of alkaloids in the Combretaceae were largely unsubstantiated. Ogan (1972) reported isolating betaine from C. micranthum, a popular West African herb known locally as kinkeleba, and simple indole alkaloids from Guiera senegalensis. However there were no further reports to verify these claims until the recent isolation of beta-carboline alkaloids from Guiera senegalensis by Fiot et al. (2006). The presence of alkaloids in the main genera of Combretum and Terminalia has yet to be confirmed. However imino sugars (polyhydroxyalkaloids (PHA) or aza sugars) are present in several species of Combretum and Terminalia from southern Africa (Katerere and Nash, unpublished). These compounds are of interest because of their potent glycosidase inhibitory activity which make them potentially useful in many carbohydratemediated disorders such as diabetes, HIV/AIDS, hepatitis B and C and cancers (Durantel et al., 2007; Nakagawa et al., 2007). Nojirimycin was the first imino sugar to be isolated in Japan in the 1960s followed by deoxynojirimicin (DNJ) which was isolated from mulberry (Moraceae) (Carroll and Nash, 2005). Synthetic templates of these compounds exhibiting fewer side effects are now becoming pharmaceutically available for diabetes, enzyme deficiencies and immunotherapy. DNJ and swainsonine-like compounds were evident in several species of Combretum (C. imberbe, C. kirkii, C. eleagnoides and C. obovatum) and Terminalia brachystegia (Katerere and Nash, unpublished). The presence of these water-soluble components may justify the herbal use of aqueous decoctions and infusions not only in the medicinal application of Combretaceae but also of many other indigenous traditional medicines. Research on water-soluble compounds is generally still in its infancy particularly in Africa due to lack of expertise. 2.5. Occurrence and chemotaxonomy of Combretaceae in southern Africa The infra generic relationship between the different Combretum taxa occurring in southern Africa (Carr, 1988) is shown in Table 1. Some taxonomic difficulties have been experienced in this field. C. collinum has been divided into ten different subspecies. Rogers and Coombes (1999) could show that subspecies of C. collinum could be differentiated by the chemical composition of the trichome secretions. Carr and Rogers (1987) investigated the use of Fig. 1. Chromatogram of acetone leaf extracts of different Combretum spp separated by thin layer chromatography using benzene–ethyl acetate–ammonia visualized with vanillin sulphuric acid. The different species are arranged according to the sectional division of Carr (1988). See Table 1. J.N. Eloff et al. / Journal of Ethnopharmacology 119 (2008) 686–699 thin layer chromatography of polar extracts to identify Combretum species. Separation of non-polar components of acetone leaf extracts of different Combretum species indicates the potential use of TLC in the chemotaxonomy of the sectional division of Combretum species (Fig. 1) (Eloff, unpublished). In the latest treatment C. paniculatum is considered a synonym of C. microphyllum based on morphology (Klopper et al., 2006). The growth forms of these two species vary tremendously growing under the same conditions in the Lowveld National Botanical Garden and the chemistry of the non-polar fractions appear to differ (numbers 12 and 19 in Fig. 1). It may be useful to follow-up this approach with other species in the Combretaceae. 3. Biological activity 3.1. Introduction to the Combretaceae Several species of the Combretaceae are used medicinally in many continents in the world. Traditional healers in Africa have used Combretum and Terminalia species for many applications including treating abdominal disorders, backache, bacterial infections, bilharzia, cancer, chest coughs, cleansing the urinary system, colds, conjunctivitis, constipation, diarrhoea, dysentery, dysmenorrhoea, earache, fever, gastric ulcers, general weakness, gonorrhoea, headaches, heart diseases, hookworm, hypertension, jaundice, leprosy, nosebleeds, oedema, pneumonia, skin diseases, sore throats, stomach and gastric problems, swelling caused by mumps, syphilis, toothache, venereal diseases (Oliver-Bever, 1986; Iwu, 1993; Hutchings et al., 1996; Neuwinger, 1996; Fyhrquist et al., 2002). Several of these uses point towards possible anti-infective activity, including antibacterial, antifungal and antiparasitic activity. In some cases bark or roots are used traditionally but based on sustainable utilization and collection of plant material from botanical gardens especially the Lowveld National Botanical Garden in Nelspruit Mpumulanga we decided to investigate only leaf material. 3.2. Bioassays used for antimicrobial investigations of Combretaceae For the quantitative determination of antibacterial activity, broth dilution methods are simple and rapid. They avoid the problems associated with agar diffusion techniques, for example difficulties in diffusion of non-polar extracts through an aqueous agar matrix. A microdilution method was developed to obtain minimal inhibitory concentration (MIC) values for plant extracts against bacteria (Eloff, 1998a). In this method, serial twofold dilutions of plant extract are prepared in wells of 96-well microtitre plates, and bacterial culture is added before incubation overnight. A tetrazolium salt is added as an indicator of bacterial growth as this salt is converted to a coloured formazan product by actively respiring cells. p-Iodonitrotetrazolium violet, or INT, at 0.2 mg/ml gave better results than other tetrazolium salts such as tetrazolium red or thiazolyl blue. MIC values are recorded as the lowest concentration resulting in a noticeable decrease in colour intensity (Eloff, 1998a). The bacteria routinely used in our laboratory include Staphylococcus aureus (ATCC 29213), Enterococcus faecalis (ATCC 29212), Pseudomonas aeruginosa (ATCC 27853) and Escherichia coli (ATCC 25922) as these species are responsible for most nosocomial diseases in hospitals (Sacho and Schoub, 1993). These specific strains were recommended for antibacterial screening purposes (NCCLS, 1990). The method distinguishes between microcidal and microstatic effects and can be used with aqueous and non-aqueous 691 extracts. The latter can be dissolved in acetone, the least toxic organic solvent to bacteria and fungi (Eloff et al., 2007). On investigating activity of a Combretum molle extract against S. aureus, the technique was 32 times more sensitive than agar diffusion techniques (Eloff, 1998a). This method is used both for screening plant extracts for antimicrobial activity and for the bioassay-guided isolation of antimicrobial compounds from plants. The technique has also been used to determine MIC values of plant extracts against fungi (Masoko et al., 2005). For qualitative detection of the number of bioactive compounds present in an extract, bioautography is a useful technique. The method we prefer is based on that of Begue and Kline (1972) and involves separating plant extract constituents by thin layer chromatography and allowing the eluent to evaporate completely from the plate. The chromatogram is sprayed with a concentrated, actively growing microbial suspension and after overnight incubation, the chromatogram is sprayed with tetrazolium violet. Where microbes are actively growing, this is indicated by red-purple areas and inhibition can be seen by the presence of clear areas. By adding carbon dioxide and by including anaerocult the method also worked well with microaerophilic/anaerobic bacteria (Beukes, 2004). The bioautography technique is more difficult with fungi because they grow more slowly and contamination can be problematic. A technique that works well with several fungi including C. albicans has been developed (Masoko and Eloff, 2005). Alternative TLC techniques are also suited to determining antioxidant activity using the free radical DPPH (1,1-diphenyl-2-picryl-hydrazyl) as a spray reagent, as evidenced with several South African Combretum and Terminalia species (Kgatle, 2007; Masoko and Eloff, 2007). Depending on the criteria for undertaking a particular investigation, bioautography is a very useful technique for deciding which of a number of plant species would be the most productive to investigate further, whether the focus is on antibacterial, antifungal or antioxidant activities (Masoko and Eloff, 2006). The method is also a practical and simple way of assessing the location of bioactive compounds as well as the effectiveness of a separation technique during bioassay-guided fractionation for the isolation of active compounds in an extract. Expression of antimicrobial activity of a plant extract or isolated compound is commonly given as an MIC when the broth dilution method is used, but because the dilution is not taken into account, this does not give an indication of the quantity of activity present in the original plant extract. To circumvent this problem, we proposed that the quantity of material extracted from one gram of dried plant material be divided by the MIC value to give the total activity of the plant (Eloff, 2000). This value called total activity (reported in ml/g) indicates the largest volume to which the biologically active compounds in one gram of plant material can be diluted and still inhibit the growth of bacteria. This method of recording results, taking into account the mass of the active fraction, facilitates the comparison of results by different researchers and would be particularly useful in investigating the most promising plants to use in rural areas for traditional healthcare (Eloff, 2000). In another application, this technique can also be valuable in bioassay-directed fractionation. The total activity or activity volume of a fraction can be expressed in ml per fraction by dividing the mass of the dried fraction in mg by the MIC in mg/ml. This number indicates to what volume the fraction of interest can be diluted and still inhibit growth of the test organism. This approach allows the rapid detection of any loss of activity and it ensures that minor biologically active compounds are not isolated in the mistaken belief that they are major active components (Eloff, 2004). The same approach can be used for isolating other biologically active compounds. 692 J.N. Eloff et al. / Journal of Ethnopharmacology 119 (2008) 686–699 Fig. 2. MIC in mg/ml of acetone leaf extracts of C. erythrophyllum plants collected over a period of 90 years in the Pretoria National Herbarium. Group on the left hand collected from Pretoria area, group on the right hand collected from different areas (Eloff, 1999). 3.3. Factors affecting biological activity of the Combretaceae The decision whether to use fresh or dried plant material to prepare extracts for laboratory studies may be influenced by various factors, such as length of time between collection of the plant material and further analysis. Many scientists prefer to use dry material to avoid problems such as fungal contamination of the plant. In a preliminary study to analyze the effects of the drying process on the antibacterial activity of Combretum erythrophyllum leaves, we found that drying by different mechanisms has a significant influence on bioactivity. Freeze drying the leaves resulted in lower activity than other drying procedures, possibly owing to the loss of volatile antibacterial compounds. Slow drying at −20 ◦ C yielded the highest antibacterial activity (Eloff and Angeh, unpublished). Stability is an important issue, as the conditions under which the plant material is stored may also affect the activity and chemical constituents of plant material. After investigating C. erythrophyllum collected in the same area and stored in herbaria for as long as 92 years, Eloff (1999a) found that the leaves did not lose antibacterial activity and the chemical composition was similar to that of recently collected, dried and extracted material. In the same study leaves collected from different areas had a wide variation in activity (Fig. 2). Storage of the prepared extracts themselves is also an issue in bioactivity studies. Acetone extracts of some tested Combretaceae species retained antibacterial and anti-inflammatory activity over prolonged periods even when stored as an acetone extract at room temperature (Eloff et al., 2001). This may be caused not only by inherent stability of the antimicrobial compounds but also by a stabilizing effect of other compounds present in the crude extracts (Eloff, 1999b; Masoko et al., 2005). In storing acetone extracts even at low temperatures, acetone may be lost through the stopper (Eloff, 2003). 3.4. Extraction of plant material The type of solvent used to prepare extracts may have an effect on the nature of the compounds extracted, and the resulting bioactivity of the extract. A series of solvents of varying polarity (methylene dichloride, acetone, ethanol, methanol, methanol/chloroform/water and water) were used to extract dried, powdered C. erythrophyllum leaves in a 1:10 ratio of dry material to solvent (Eloff, 1998b), to ascertain the value of each extractant. Several parameters, including the rate of extraction, the quantity extracted, the diversity of compounds extracted, the diversity of inhibitory compounds extracted, the ease of subsequent handling of the extracts, toxicity of the solvent in the bioassay process, and the potential health hazard of the extractants were evaluated. The solvents used in the study were compared by allocation of points on an arbitrary five point weighted scale, and acetone achieved the best results (Eloff, 1998b). In investigation of many species using different extractants subsequently, acetone was always one of the best extractants (Kotze and Eloff, 2002). The efficiency of extraction has to be optimized to ensure that as many of the potentially active constituents as possible are extracted. The method of extraction developed in our laboratory involves grinding dried leaves finely (to a particle diameter of approximately 10 ␮m) and shaking at a rapid rate on a shaking machine for three 5-min periods. Using this method, a higher percentage of C. erythrophyllum dry mass was extracted (Eloff, 1998b), than the value obtained after 24 h of extraction on a shaking machine with more coarsely ground plant material. Different extractants can be used to simplify extracts prior to isolation of antibacterial compounds from plants. Combretum microphyllum was used to prepare extracts of dried ground leaves using solvents with different polarities, including hexane, carbon tetrachloride, di-isopropylether, ethyl ether, methylene dichloride, tetrahydrofuran, acetone, ethanol, ethyl acetate, methanol and water (Kotze and Eloff, 2002). Thin layer chromatography (TLC) and a microplate serial dilution method were used to determine chemical composition and antibacterial activity against Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli and Enterococcus faecalis of extracts, respectively. The intermediate polarity solvents di-isopropyl ether, ethanol, ethyl ether, acetone and ethyl acetate yielded extracts with a high antibacterial activity with a lower quantity of other non-active compounds and appear to be useful for isolating bioactive compounds from Combretum leaves. Extractants can also be used to selectively remove bulky, inactive constituents from plant material to result in extracts with much improved bioactivity compared to the initial crude extract. This technique allows the advantage of potential additive or synergistic interactions between active principles with different mechanisms of action in the extract. The concept was successfully used to produce an extract from Combretum woodii with high antibacterial and antioxidant activity (Eloff et al., 2005a, 2006; Zishiri, 2005). 3.5. Antibacterial activity 3.5.1. Initial work: Combretum erythrophyllum Our investigations of the Combretaceae family began with C. erythrophyllum. In initial work on acetone leaf extracts, liquid/liquid fractionation produced a better group separation of compounds than solid phase extraction on normal or reversed phase silica gel (Martini and Eloff, 1998). Bioautography showed that there were at least 14 different inhibitors of widely differing polarity present active against S. aureus in the original plant extract. Interestingly, this study showed that water did not extract inhibiting compounds from the dried ground leaves, possibly because the inhibiting compounds were made unavailable by lipid soluble membranes. Extraction with acetone may have released several water-soluble antimicrobials. Martini (2002) concluded that the large number of antimicrobial components may explain why Combretum is so widely used for medicinal purposes throughout Africa. Evaluating the traditional method of preparation of the medicinal extract may be of value in substantiating this statement, as if a decoction is prepared, heat may act in a similar manner to the organic solvent in disrupting cell membranes and increasing availability of non-polar plant compounds. 3.5.2. Screening of southern African Combretaceae species for antibacterial activity Following the promising results obtained with Combretum erythrophyllum, other members of the Combretaceae were screened J.N. Eloff et al. / Journal of Ethnopharmacology 119 (2008) 686–699 for antibacterial activity to discover the best species for isolation of active compounds (Eloff, 1999b). Leaves of 27 species of Combretum, Terminalia, Pteleopsis and Quisqualis were dried, powdered and extracted with acetone. The MIC values of extracts were determined by the microplate serial dilution technique using Staphylococcus aureus, Enterococcus faecalis, Pseudomonas aeruginosa and Escherichia coli as test organisms. Interestingly, all the extracts inhibited the growth of the four bacteria, with MIC values between 0.1 and 6 mg/ml, and an average MIC of 2.01 mg/ml. The Gram-positive strains were slightly more sensitive than the Gramnegative strains. With regard to the MIC values and the total content in each plant, the seven species with the highest antibacterial activity were C. molle, C. petrophilum, C. moggii, C. erythrophyllum, C. padoides, C. paniculatum and C. mossambicense (Eloff, 1999b). 3.5.3. Combretum woodii The best extractant for isolation and characterization of antibacterial compounds was analyzed with respect to Combretum woodii (Eloff et al., 2005a). Dried ground leaves were extracted with 10 different solvents (hexane, di-isopropyl ether, diethyl ether, methylene dichloride, ethyl acetate, tetrahydrofuran, acetone, ethanol, methanol and water). All the extracts had antibacterial activity except for the water extract, which had no activity. Intermediate polarity solvents extracted about 10% of the dry mass compared to about 3% with the more polar or non-polar solvents. Extracts prepared using these solvents additionally had higher antibacterial activity than more polar or non-polar extractants (Eloff et al., 2005a). Ethyl acetate proved to be the best extractant with an average MIC value of 80 ␮g/ml for the four pathogens (Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli and Enterococcus faecalis) followed by acetone, and methylene dichloride, both with MICs of 140 ␮g/ml. When considering the quantity extracted from the powdered leaf material, the total activity was highest for methylene dichloride (1309 ml/g) followed by acetone (1279 ml/g) extracts. The Rf values of the antibacterial compounds shown in bioautography indicated that the antibacterial compound was not a polyphenol or a tannin (Eloff et al., 2005a). These results provided impetus for the isolation and characterization of antibacterial compounds from the extracts. In continuing work, the stilbene combretastatin B5 was isolated from the acetone extract of leaves of C. woodii (Eloff et al., 2005b). This compound showed significant activity against S. aureus (MIC = 16 ␮g/ml), but had lower activity against P. aeruginosa and E. faecalis (MIC values against both of 125 ␮g/ml) and only slight activity against E. coli. Interestingly, the concentration of combretastatin B5 in the leaves was relatively high, around 5–10 mg/g. In an MSc study aiming to develop an extract that could be used as an alternative feed additive in poultry production, further studies were conducted on Combretum woodii (Zishiri, 2005). The potentised extract should preferably be rich in antibacterial activity to control proliferation of undesired microorganisms, and also should possess antioxidant activity to boost the immune system of the poultry. An extract was developed by pretreatment with a single direct extraction with hexane prior to extraction with acetone. The trolox equivalent antioxidant activity capacity (TEAC) value of this extract determined using the method of Re et al. (1999) was 2.3, representing an increase in TEAC value of 283% compared to the value of 0.83 of the crude acetone extract. The average MIC of the crude acetone extract against S. aureus, P. aeruginosa, E. coli and E. faecalis decreased from 150 to 80 ␮g/ml in the optimal extract, reflecting an improvement in antibacterial activity of 87.5%. The optimal extract was effective against the poultry pathogens Campylobacter jejuni and Clostridium perfringens with MIC values ranging from 40 to 80 ␮g/ml. It was also active against multi-resistant strains of 693 E. coli and Salmonella enteritidis (MIC values of 125 ␮g/ml for both strains). The in vitro toxicity of the optimized extract was ascertained using the brine shrimp assay and the MTT cytotoxicity assay on Vero monkey kidney cells. LC50 results from the brine shrimp assay and the MTT cytotoxicity assay on monkey kidney cells gave values of 863 and 226 ␮g/ml, respectively indicating low toxicity. These results implied that although in some cases the MICs of the optimal extract were higher than those of typical antibiotics, owing to its relatively low toxicity, large quantities of the extract may possibly be fed to achieve the desired activity without causing any toxicity in the poultry (Zishiri, 2005). The major antioxidant compound from Combretum woodii was isolated by silica gel column chromatography and identified as combretastatin B5 (Zishiri, 2005). This compound was previously shown to be the major antibacterial compound in C. woodii leaves (Famakin, 2002). Combretastatin B5 (CB5) was cytotoxic in vitro in the MTT assay on monkey kidney cells with an LC50 value of 10 ␮g/ml. In vitro cytotoxicity of CB5 could be due to its antimitotic activity. The TEAC value of 7.9 reported by Zishiri (2005) indicates that combretastatin B5 has about eight times the antioxidant capacity of vitamin E. This was the first report of the antioxidant activity of any of the combretastatins. A remarkable aspect of potentising the C. woodii extract is that the therapeutic index (ratio of the amount of a therapeutic agent that causes the therapeutic effect to the amount that causes a toxic effect) increased from 0.6 for the pure compound to 5.6 for the potentised extract. This indicates that other compounds present in the extract protected the monkey kidney cells against the toxic effect (Eloff et al., unpublished). Replacing antibiotic feed additives widely used in poultry production is an important area of research because the European Union has prohibited the importation of poultry if antibiotic feed additives are used in the production. In an in vivo study (Zishiri, 2005), tolerance of broiler chickens to the optimal extract was assessed at doses of 2, 5 and 10 mg/kg. After 21 days of dosing with the optimal extract in the feed, none of the chickens died or showed any behavioural signs of toxicity. There were no statistically significant differences in weight gain between broilers fed the optimal extract and the positive and negative control. There was also no positive correlation between weight gain and amount of the optimal extract incorporated in feed. Even though the optimal extract did not cause significant growth promotion relative to the positive and negative control, the 2 mg/kg dose showed the best feed conversion ratio (FCR), with a 6.2% improvement compared to the negative control. The positive control was the only other feed regimen to provide a positive FCR with an improvement of 1.73% compared to the negative control. Feed purchasing may represent up to 80% of the costs of broiler production, so this is an important finding. If these results can be confirmed, the product may therefore have commercial value. Repetition of the experiment with lower doses of the optimal extract on poultry challenged with bacterial infections is needed to confirm the commercial applicability of this product (Zishiri, 2005). 3.5.4. Antibacterial activity of other members of the Combretaceae The use of different extractants to extract leaf material of Combretum microphyllum was examined (Kotze and Eloff, 2002). It was found that there was a surprising similarity in the chemical composition of the non-polar components of extracts using extractants of widely varying polarity, and belonging to different selectivity groups. Kotze and Eloff (2002) explained this by stating that there may be large quantities of saponins or other soap-like compounds that would solubilise very non-polar compounds into polar 694 J.N. Eloff et al. / Journal of Ethnopharmacology 119 (2008) 686–699 extractants. Sodium bicarbonate did not appear to work well as an extractant for C. microphyllum. The lowest MIC value was obtained using acetone on intact leaves. Di-isopropyl ether, ethanol, ethyl ether, acetone and ethyl acetate extracted constituents of high antibacterial activity with a lower quantity of other non-active compounds, and this was held to be a useful property for isolating bioactive compounds. Five antibacterial triterpenoids were isolated from Combretum imberbe leaves, with levels of antibacterial activity MIC values against Staphylococcus aureus and Escherichia coli ranging from 16 to 62 ␮g/ml (Angeh, 2005) (Table 3). It was concluded that although the major antibacterial compounds were isolated, the antibacterial activity of the crude extract was higher than could be extrapolated from the activity of the isolated compounds, and this was attributed to synergistic effects. Some of the compounds also had reasonable anti-inflammatory activity, as demonstrated by inhibition of 3␣hydroxysteroid dehydrogenase activity (Angeh, 2005). This lends support to an earlier suggestion, following a preliminary screening investigation, that the same compounds in Combretum species may be responsible for antibacterial and anti-inflammatory activity (Eloff et al., 2001). In research on other species, two antibacterial flavonoids were isolated from Combretum apiculatum subsp. apiculatum (Serage, 2004). Three antibacterial compounds, a new oleanene-type triterpenoid glycoside and two known triterpenoids, were also isolated from Combretum padoides (Angeh et al., 2007b). The compounds were not cytotoxic, indicating that pentacyclic triterpenes from C. padoides have selective antibacterial activity with no cytotoxic activity. Rabie (2005) could show that extracts of Quisqualis littorea and Pteleopsis myrtifolia had antibacterial activity against common nosocomial bacteria with MIC from 0.04 to 0.32 mg/ml. Some of these extracts inhibited the growth of cancer cell lines MCF-12A, H157, WHC03 and HeLa. She isolated taraxerol from Pteleopsis myrtifolia and it had a MIC values of 0.04 and 0.016 mg/ml against Escherichia coli and Enterococcus faecalis, respectively. 3.6. Antifungal activity New antifungal drugs associated with different mechanisms of action to currently available drugs, or with lower toxicity and lower cost, are urgently needed. Our focus is on fungal pathogens, such as Candida albicans and Cryptococcus neoformans that afflict immunocompromised patients. This may be as a result of the increasing occurrence of opportunistic mycotic infections associated with AIDS, or infections developing after treatment with immunosuppressive drugs for example. The antifungal activity of Combretum and Terminalia species was investigated using a slightly different version of the antibacterial microdilution technique described above. Based on the observation that fungal growth was less predictable than that of the bacteria, Masoko et al. (2005) modified the microdilution method of Eloff (1998a) by adding INT prior to incubation of fungi together with plant extract so that it was possible to note when sufficient growth had taken place to read results. Fortunately it was found that acetone, similar to the case with bacteria, is not toxic to the fungi at the concentrations used in the assay (Eloff et al., 2007). Acetone had an MIC of 51% for several fungi and was less toxic than ethanol, methanol, or dimethylsulphoxide to the fungi. Organic extracts prepared with hexane, dichloromethane and methanol could be therefore be redissolved in acetone for use in the assay. Masoko et al. (2005) detected noteworthy antifungal activity of several Terminalia species (Combretaceae) against several species of fungal organisms. Fungi isolated from clinical cases of disease in animals, were used in the screening procedure and included Candida albi- cans, Cryptococcus neoformans, Aspergillus fumigatus, Microsporum canis and Sporothrix schenkii. These fungi represent the different morphological forms of fungi, that is, yeasts (Candida albicans and Cryptococcus neoformans), thermally dimorphic fungi (Sporothrix schenckii) and moulds (Aspergillus fumigatus). They are the most commonly encountered and important disease-causing fungi of animals. In this initial work, acetone, hexane, dichloromethane and methanol leaf extracts prepared from six Terminalia species (T. prunioides, T. brachystemma, T. sericea, T. gazensis, T. mollis and T. sambesiaca) were tested for antifungal efficacy (Masoko et al., 2005). Methanol extracted the highest quantity of material, but the acetone extracts had the highest antifungal activity. Some extracts had MIC values as low as 0.02 mg/ml, and T. sericea extracts were the most active against nearly all microorganisms tested. Acetone and methanol extracts of six Terminalia species exhibited zones of antioxidant activity after spraying developed TLC chromatograms with DPPH (Masoko et al., 2005). However, hexane and dichloromethane extracts did not have any antioxidant activity. It appeared that the antioxidant compounds were all relatively polar in each species as they were generally visible on the lower half of the TLC plate. In further work on these Terminalia species (Masoko and Eloff, 2005), direct bioautography using yeast and mould species was used to confirm that the compounds responsible for antifungal activity in these species were non-polar, and were not tannins. The bioautography method was modified from that used with bacterial species. Actively growing cultures on Sabouraud dextrose agar were collected by light swabbing and transferred to sterile fresh Sabouraud broth. After incubation in broth for an hour, developed TLC plates were sprayed with this culture. Owing to the variable growth rates of the fungal species used, problems were encountered with overgrowth of mycelia and disappearance of the zones of inhibition. To overcome this, the chromatograms were sprayed with INT three to four hours after spraying with the test fungi, enabling rapid identification of positive results as they became visible (Masoko and Eloff, 2005). The bioautography results showing several active compounds in Terminalia species (Masoko and Eloff, 2005) correlated with the MIC values reported in the previous study (Masoko et al., 2005). Bioautography is a useful tool as it enables the dereplication of active compound isolation from related taxa. Bioautography was also used to analyze antifungal compounds in hexane, dichloromethane, acetone and methanol extracts of 24 species belonging to the genus Combretum (Masoko and Eloff, 2006). In general, acetone extracted the most antifungal compounds from the plant material. Cryptococcus neoformans was the most susceptible organism to the extracts, and Aspergillus fumigatus was the most resistant. Similarly to the Terminalia extracts (Masoko and Eloff, 2005), it was concluded that, based on the Rf values of the antifungal compounds extracted, activity of these species was not only attributable to tannins in Combretum species. Upon comparing results obtained for species in the different sections of the genus, it was determined that there is a correlation between the taxonomic classification and the number of antifungal compounds. Subsequent to the bioautography study, antifungal MIC values were determined for acetone, hexane, dichloromethane and methanol leaf extracts of the same 24 Combretum species against the same 5 fungal pathogens (Masoko et al., 2007). Microsporum canis was the most sensitive fungal species, and Aspergillus fumigatus the most resistant. Methanol generally extracted the largest quantity of material from the plants than any of the other solvents. The acetone extracts however, had the highest antifungal activity, with MIC values as low as 0.02 mg/ml. Extracts of C. nelsonii had low average MIC values and high total activity values, followed by C. albopunctatum and C. imberbe. Terminolic acid, asiatic acid and arjunolic acid were isolated from C. nelsonii and a mixture of Table 3 Compounds isolated by bioassay-guided fractionation from Combretaceae members and biological activity in ␮g/ml Plant Compound isolated S.a E.f P.a E.c Toxicity LD50 Other activities Reference C. apiculatum subsp. apiculatum Flavokawain Alpenitin Pinocembrin Terminoic acid 40 40 80 330 400 40 60 nd 300 130 300 nd 600 250 130 nd nd nd nd nd nd nd nd In vivo activity Serage (2004) Serage (2004) Serage (2004) Kruger (2004) 5-Hydroxy-7,4′ -dimethoxyflavone Quercitin 5,3′ -dimethylether Rhamnazin Rhamnocitrin Genkwanin Apigenin Kaempferol Combretastatin B5 Combretastatin B5 >100 >100 >100 50 >100 nd nd 16 nd 50 38 25 38 75 nd nd 125 nd 100 100 100 100 100 nd nd 125 nd 75 75 100 75 100 nd nd 250 nd LDH low LDH low LDH low LDH low LDH low nd nd nd 10 ␮g/mla V. cholera 38 V. cholera 50 V. cholera 50 V. cholera 50 V. cholera 50 nd nd nd TEAC = 7.9 Martini et al. (2004) Martini et al. (2004) Martini et al. (2004) Martini et al. (2004) Martini et al. (2004) Martini et al. (2004) Martini et al. (2004) Eloff et al. (2005b) Zishiri (2005) Apigenin Taraxerol Mixture asiatic acid and arjunolic acid 1,3-Dihydroxy-12-oleanen-29-oic 1-Hydroxy-12-olean-30-oic acid 3,30-Dihydroxyl-12-oleanen-22-one 1,3,24-Trihydroxyl-12-olean-29-oic acid 1,23-Dihydroxy-12-oleanen-29-oic acid-3-O-2,4-di-acetyl-lrhamnopyranoside 1␣,23␤-Dihydroxy-12-oleanen-29-oicacid-23␤-O-␣-4acetylrhamnopyranoside 1,22-Dihydroxy-12-oleanen-30-oic acid Ethylcholesta-7,22,25-trien-O-␤-dglucopyranoside 160 40 nd 125 94 125 63 320 16 nd 125 [24 125 >250 >320 630 nd >250 >250 >250 >250 320 40 nd 16 >250 16 16 nd nd nd 34.9 10.5 nd Anticancer Antifungal** Anticancer MIC 0.2–1.6 ␮g/ml 5 fungi Anti-inflammatory activity Determined 47.3 Komape (2005) Rabie (2005) Masoko (2006) Angeh et al. (2007a) Angeh et al. (2007a) Angeh et al. (2007a) Angeh et al. (2007a) 63 >250 >250 16 17.5 Angeh et al. (2007a) New compound 63 >250 63 >250 >50 Angeh et al. (2007b) New compound 31 125 63 >250 44.7 Angeh et al. (2007b) >250 >250 63 >250 >50 Angeh et al. (2007b) T. sericea C. woodii C. woodii C. vendae Pt. myrtifolia C. nicholsonii C. imberbe C. padoides Anticancer In animal experiment extract was better than gentamicin Developing extracts with high activity, also animal productivity J.N. Eloff et al. / Journal of Ethnopharmacology 119 (2008) 686–699 C. erythrophyllum Note Bacteria: S.a, Staphyloccoccus aureus; E.f, Enterococcus aureus; P.a, Pseudomonas aeruginosa; E.c, Escherichia coli. ** Hela cells. a Cytotoxicity LD50 MTT assay Vero kidney cell. 695 696 J.N. Eloff et al. / Journal of Ethnopharmacology 119 (2008) 686–699 the last two had excellent activity against several fungi (Masoko, 2006). 3.7. Other in vitro biological activities 3.7.1. Anti-inflammatory activity, anthelmintic, antischistosomal and DNA-damaging activity One of the traditional medicinal uses of Combretum species involves treating pain and this may indicate that extracts possibly have anti-inflammatory properties. The anti-inflammatory activity and stability of 20 Combretum species growing under the same environmental conditions was compared, and it was found that all the extracts had some anti-inflammatory activity with average 65% inhibition of cyclooxygenase-1 activity (Eloff et al., 2001). The inhibition was surprisingly stable with no loss of activity after storage for 3 months at room temperature. There was a reasonably good correlation between total anti-inflammatory activity and total antibacterial activity of the same taxa studied earlier. As was alluded to earlier in the discussion of Combretum imberbe, Eloff et al. (2001) suggested that similar compounds may be responsible for these biological activities, especially since in both cases the bioactivity is stable. In a broad screening procedure, leaf extracts of 20 Combretum species were screened for anti-inflammatory, anthelmintic, antischistosomal (anti-bilharzia) and DNA-damaging activity (McGaw et al., 2001). Ethyl acetate extracts were the most active, followed by acetone and then water extracts. Some species, including C. apiculatum, C. hereroense, C. molle and C. mossambicense had substantial activity in more than one bioassay. C. apiculatum in particular was very active in the anti-inflammatory, anthelmintic and DNA-damage tests. Extracts of different polarities were effective, implying that active compounds possessed a wide range of polarities. 3.7.2. Antioxidant activity Many plant extracts have been shown to possess antioxidant properties, and various investigations have shown that these antioxidant compounds may also possess other activities such as anti-inflammatory, antitumour, antimutagenic, anticarcinogenic, antibacterial or antiviral activities. In a study investigating the antioxidant potential of 24 Combretum and Terminalia species, leaf extracts were prepared with acetone, hexane, dichloromethane and methanol (Masoko and Eloff, 2007). TLC plates were developed and sprayed with 0.2% DPPH in methanol for antioxidant screening. Visualization of separated bands exhibiting antioxidant activities enabled the localization of the active compounds. Acetone and methanol extracts showed antioxidant activity, but hexane and dichloromethane extracts did not have any antioxidant effects. C. hereroense had the highest number of active compounds, followed by C. collinum ssp. taborense with 16 and 10 antioxidant constituents, respectively. All Terminalia species extracted with acetone and methanol had compounds with antioxidant activity. T. gazensis and T. mollis methanol extracts had 11 and 14 active compounds respectively in one of the solvent systems used (Masoko and Eloff, 2007). Methanol extracts of leaves of 10 different Combretum species were evaluated for qualitative antioxidant activity. Leaf extracts of Combretum apiculatum subsp. apiculatum had the most antioxidant compounds. Antioxidant-directed fractionation of the leaf extracts of C. apiculatum led to the isolation of four antioxidant compounds from ethyl acetate and butanol soluble fractions. The structures of the compounds were determined by spectral analyses (1 H NMR, 13 C NMR and MS) and identified as cardamomin (1), pinocembrin (2), quercetrin (3) and kaempferol (4) (Kgatle, 2007). These compounds occur commonly in plant extracts, but the anti-oxidant activities of all these compounds were not known previously. In a quantitative antioxidant assay using DPPH with l-ascorbic acid as positive control, the more polar fractions (ethyl acetate and butanol) obtained by solvent–solvent fractionation had the highest activity among the extracts with EC50 values of 3.91 ± 0.02 and 2.44 ± 0.02 ␮g/ml, respectively. Of the isolated compounds, quercetrin (3) and kaempferol (4) had strong antioxidant activity with EC50 values of 11.81 ± 85 and 47.36 ± 0.03 ␮M, respectively. Cardamomin (1) and pinocembrin (2) did not have strong activity as these compounds could not scavenge 50% of the DPPH radical at the highest concentration (200 ␮M) tested. l-Ascorbic acid was used as a positive control and standard (EC50 = 13.37 ± 0.20 ␮M or 2.35 ␮g/ml). The antioxidant activity of the isolated compounds supported structure–activity relationships developed by other authors. The cytotoxicity of cardamonin and pinocembrin was evaluated using the MTT assay, with berberine as positive control and DMSO as negative control. At higher concentrations than 50 ␮g/ml of cardamomin or pinocembrin the cells were not viable. Cardamomin was more toxic to the cells (LC50 of 1.97 ␮g/ml) than pinocembrin (LC50 of 29.47 ␮g/ml) and even the positive control, berberine (LC50 of 12.35 ␮g/ml) (Kgatle, 2007). 3.7.3. Antiviral activity In a PhD study, Samdumu (2007) discovered that acetone and water extracts of Combretum paniculatum had good antiviral efficacy, reducing the cytopathic effect of feline herpesvirus by 3 log10 . Cytotoxicity against Vero cells was observed only at relatively high concentrations. Nine compounds with broad spectrum antimicrobial activity were isolated from C. paniculatum and were identified as cholest-5-en-3-ol, 2-phyten-1-ol, isoquercitrin, p-coumaric acid, 2,3,8-tri-O-methylellagic acid, beta-sitosterol, gallocatechin, apigenin and apigenin-7-glucoside. Several of these compounds were active against bacteria as well as fungi. The compounds however had no antiviral activity against coxsackievirus strain B3 Nancy, influenza virus type A strain Hong Kong and herpes simplex virus type 1 strain K1 (Samdumu, 2007). The constituents responsible for antiviral efficacy are yet to be isolated from this species. 3.8. Toxicity testing We routinely use in vitro bioassays to determine potential toxicity of plant extracts and compounds isolated from the extracts. This supplies an indication of the selective biological activity of the test substance. The brine shrimp larval lethality assay is a rapid and simple test for cytotoxicity, and may give a useful preliminary indication of toxicity. However, there are limitations to the conclusions that can be drawn regarding activity in this assay. Using the method of Solís et al. (1993), larvae are hatched in artificial seawater and incubated with varying concentrations of test solution for 24 h. The percentage mortality, or LD50 values, may then be calculated. Cell line cytotoxicity assays with the African Green monkey kidney (Vero) cell line are used to calculate cytotoxicity of extracts and compounds. The MTT [3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide] assay (Mosmann, 1983) is based on the ability of a mitochondrial dehydrogenase enzyme from viable cells to cleave the tetrazolium rings of a yellow MTT solution to form dark blue formazan crystals which are largely impermeable to cell membranes, thus resulting in their accumulation within healthy cells. The aqueous growth medium is aspirated from the cells, and DMSO used to solubilise the formazan crystals. The number of surviving cells is directly proportional to the level of the formazan product created. The colour can then be quantified using a spectrophotometer (Mosmann, 1983), and LC50 values calculated for each test substance. With this method, it is crucial to keep in mind that certain substances may react with MTT to cause a colour J.N. Eloff et al. / Journal of Ethnopharmacology 119 (2008) 686–699 change in the absence of cells, particularly in the case of antioxidants (Bruggisser et al., 2002; Shoemaker et al., 2004). Microscopic visualization of the cells prior to addition of the MTT can provide an indication of cytopathic effect on the cells. Other assays of cell viability are also available and a combination of these can be used to achieve a more realistic idea of the cytotoxicity of the test materials. In the brine shrimp assay, C. nelsonii, C. imberbe, C. albopuntactum and T. sericea acetone extracts were relatively non-toxic, with LC50 values of 3.16, 2.30, 3.05 and 2.43 mg/ml, respectively (Masoko, 2006). Using the cell line MTT assay, the LC50 values were 75.7, 168.6, 121.7 and 102.9 ␮g/ml for C. nelsonii, C. imberbe, C. albopunctactum and T. sericea, respectively (Masoko, 2006). 3.9. In vivo activity of Combretaceae extracts and isolated compounds There are unavoidable limitations associated with in vitro tests, such as the lack of a physiologically representative environment and absence of metabolic activation. Therefore it is necessary to confirm promising efficacy observed in laboratory tests with activity in an animal model to proceed with the development of a potential new antibacterial or antifungal preparation. A method was developed and approved by the appropriate ethical committee to test crude extracts of Terminalia sericea and a compound isolated from the plant, terminoic acid, for in vivo topical antibacterial activity (Kruger, 2004). The procedure was also used by Masoko (2006) to detect topical antifungal activity. In this method, extracts and compounds are mixed with aqueous cream in defined percentages and applied to lesions made on rat skin. Where the lesions are not infected with a bacterial or fungal pathogen, wound-healing activity is measured, but where inoculation of the wound is performed with a pathogen, potential antibacterial or antifungal activity is analyzed. Wound irritancy and wound healing may be assessed using macroscopical, physical and histological methods. Aspects observed include wound healing, erythema, exudate formation and possible toxic effects of the extracts. Antibacterial in vitro results of the acetone extract of T. sericea and terminoic acid against Staphylococcus aureus gave MIC values of 1.56 and 0.33 mg/ml, respectively (Kruger, 2004). In the in vivo study, lesions were created in the skin of the back of rats and infected with a pathogenic Cowan A strain of S. aureus. The wounds were then treated with either a 20% emulsified cream preparation of the acetone extract or a 1% cream of terminoic acid applied to the wounds daily. The results revealed that terminoic acid and the crude extract had a significantly higher antibacterial effect than a commercial gentamicin cream (Kruger, 2004). It was a pleasant surprise that a crude extract with a relatively low in vitro activity could yield such good in vivo results. These results support the ethnobotanical use of Terminalia sericea for the topical treatment of wounds. Similarly, the acetone leaf extracts of C. nelsonii, C. albopunctactum, C. imberbe and T. sericea possessed growth inhibitory activities against fungal pathogens in vivo (Masoko, 2006). In a study investigating the anticoccidial effects in poultry of plant extracts with known antioxidant activity, Naidoo et al. (2008) reported that orally administered Combretum woodii was toxic to the birds at a concentration of 160 mg/kg. This was the maximum concentration of a 70% acetone extract that was soluble in water. Although Zishiri (2005) concluded that relatively large quantities of the plant extract may be fed to poultry, a more accurate concentration needs to be quantified to obtain benefit from feeding the extract to poultry in the absence of the toxic effects noted by Naidoo et al. (2008). In vivo toxicity, both acute and chronic, is another important aspect to consider, as cell-based assays detecting cytotoxic effects 697 are insufficient to indicate toxic effects of ingested or topically applied medications. This and genotoxicity, is an area of future investigation in the Phytomedicine Programme. 4. Conclusions From the data presented here many follow-up studies can be identified. The original aim of this research was to investigate members of the Combretaceae for compounds with exceptional antimicrobial activity that could serve as model structures to develop new antimicrobial compounds to address the growing antimicrobial resistance experienced all over the world. The compounds isolated by bioassay-guided fractionation were mainly flavonoids, terpenoids and a bibenzyl. With the exception of the data of Masoko (2006) where the antifungal activity of a mixture of asiatic acid and arjunolic acid had activities as good as amphotericin B (MIC 0.2–0.4 ␮g/ml against C. neoformans, Microsporium canis and Sporothrix schenckii) the activities of isolated compounds were disappointingly low. On the other hand nearly all the crude acetone extracts had substantial activity. In some cases MICs were as low as 40 ␮g/ml. This shows the possibility that crude extracts could be used rather than pure compounds. The explanation for this situation is probably the occurrence of synergistic activities between different components within a crude leaf extract. The results that we have obtained confirm the ethnomedicinal use of the different species to a very large extent. The main difficulty is that at least as far as antibacterial and antifungal activity is concerned there was hardly any activity in aqueous extracts. One the one hand this shows that the criticism raised against investigating the Combretaceae that the activity is mainly due to tannins with very little potential use because biological unavailability may be flawed. On the other hand it indicates that because water is the only solvent freely available to rural people these active extracts cannot be used even though the plants grow in the environment. We have started doing some work in this area by evaluating the efficiency of dilute soap mixtures to extract active compounds from plants. The challenge from this point appears to provide products for the herbal medicine industry based on extracts where the activity has been enhanced by removing inactive compounds as has been done by Zishiri (2005) and also in another application by Irene Angeh (Angeh, 2006; Eloff et al., 2006). Research into the mechanism of synergy could also provide very interesting data. 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