McGaw_129-134.qxd INTRODUCTION Gunnera is the only genus of the family Gunnera- ceae, which includes 30–40 herbaceous species, mostly distributed in the southern hemisphere (Wanntorp, Wanntorp, Oxelman & Kãllersjö 2001). The greatest diversity is found in South and Central America. A few species are also found in Hawaii, New Zealand, Tasmania, Africa and the Malay Archipelago (Wanntorp et al. 2001). Gunnera per- pensa L. is widely distributed in the southern, east- ern and northern parts of South Africa and north- wards into tropical Africa (Van Wyk, Van Oudtshoorn & Gericke 1997). It is a vigorous perennial herb that is restricted to moist habitats and grows in marshy areas and along river banks. It has large, round, pumpkin-like leaves and fleshy, pink tuberous rhi- zomes (Van Wyk & Gericke 2000). The plant is known in South Africa by various names including ugobho (Zulu), qobo (Sotho), rambola-vhadzimu (Venda), river pumpkin (English) and rivierpampoen (Afrikaans) (Van Wyk et al. 1997). 129 Onderstepoort Journal of Veterinary Research, 72:129–134 (2005) Is the use of Gunnera perpensa extracts in endometritis related to antibacterial activity? L.J. McGAW1, R. GEHRING2, L. KATSOULIS3 and J.N. ELOFF1* ABSTRACT McGAW, L.J., GEHRING, R., KATSOULIS, L. & ELOFF, J.N. 2005. Is the use of Gunnera perpen- sa extracts in endometritis related to antibacterial activity? Onderstepoort Journal of Veterinary Research, 72:129–134 Rhizome extracts of Gunnera perpensa are used in traditional remedies in South Africa to treat endometritis both in humans and animals. An investigation was undertaken to determine whether this plant possesses antibacterial activity, which may explain its efficacy. Gunnera perpensa rhizome extracts were prepared serially with solvents of increasing polarity and tested for antibacterial activ- ity. Test bacteria included the Gram-positive Enterococcus faecalis and Staphylococcus aureus and the Gram-negative Escherichia coli and Pseudomonas aeruginosa. A moderate to weak level of anti- bacterial activity in most of the extracts resulted, with the best minimal inhibitory concentration (MIC) value of 2.61 mg ml–1 shown by the acetone extract against S. aureus. The extracts were also sub- mitted to the brine shrimp assay to detect possible toxic or pharmacological effects. All the extracts were lethal to the brine shrimp larvae at a concentration of 5 mg ml–1. The acetone extract was extremely toxic at 1 mg ml–1, with some toxicity evident at 0.1 mg ml–1. The remainder of the extracts generally displayed little activity at concentrations lower than 5 mg ml–1. In summary, the results indi- cate that although the extracts demonstrated a level of pharmacological activity, the relatively weak antibacterial activity is unlikely to justify the use of G. perpensa rhizomes in the traditional treatment of endometritis. Rather, the slightly antibacterial nature of the rhizomes may contribute to an addi- tive effect, along with their known uterotonic activity, to the overall efficacy of the preparation. Keywords: Antibacterial, brine shrimp, endometritis, ethnoveterinary, Gunnera perpensa, traditional medicine * Author to whom correspondence is to be directed. E-mail: kobus.eloff@up.ac.za 1 Programme for Phytomedicine, Department of Paraclinical Sciences, Faculty of Veterinary Science, University of Pre- toria, Private Bag X04, Onderstepoort, 0110 South Africa 2 Department of Paraclinical Sciences, Faculty of Veterinary Science, University of Pretoria, Pretoria, 0002 South Africa 3 Department of Pharmacology, University of Witwatersrand, Johannesburg, 4000 South Africa Accepted for publication 19 January 2005—Editor Infusions or decoctions of the rhizome of G. per- pensa are used by black South African women to induce or augment labour and as a postnatal med- ication to expedite the expulsion of the after-birth (Hutchings, Scott, Lewis & Cunningham 1996; Kaido, Veale, Havlik & Rama 1997; Van Wyk et al. 1997; Van Wyk & Gericke 2000). Decoctions are also administered to animals by the Zulu and south- ern Sotho to assist the expulsion of the placenta (Watt & Breyer-Brandwijk 1962). According to anec- dotal reports, this herbal remedy is used to treat cattle by subsistence farmers and also by commer- cial farmers of the Underberg region of KwaZulu- Natal, South Africa to assist in the expulsion of fetal membranes, and in the treatment and prevention of endometritis (T. Collins, unpublished data 2002). Conventional treatment of retained foetal mem- branes and endometritis in cattle includes manual removal of the membranes, stimulation of uterine contraction and stimulation of uterine defences as well as topical and systemic antibacterial therapy (Kudlác 1991). The use of G. perpensa for treating humans and animals may therefore be due to anti- bacterial effects. In a preliminary screening, McGaw, Jäger & Van Staden (2000) reported antibacterial activity of the aqueous rhizome extract against one bacterial pathogen, the Gram-positive Staphylococ- cus aureus. However, no activity was demonstrated against Bacillus subtilis (Gram-positive) or the Gram- negative bacteria E. coli and Klebsiella pneumoniae. The chemistry of Gunnera species appears not to have been thoroughly studied, although Watt & Breyer-Brandwijk (1962) mention the presence of a bitter principle, celastrin, in G. perpensa. Doyle & Scogin (1988) detected caffeic acid, ellagic acid, ellagitannins and quercetin in Gunnera densiflora and Gunnera manicata. Alkaloids, iridoids, proan- thocyanidins, saponins and sedoheptulose were not present in these species (Doyle & Scogin 1988). The compounds present in these two Gunnera spe- cies that have antibacterial activity, are caffeic acid (Thiem & Goslinska 2004), ellagic acid (Ohemeng, Schwender, Fu & Barrett 1993), ellagitannins (Nish- izawa, Nakata, Kishida, Ayer & Browne 1990) and quercetin (Basile, Sorbo, Giordano, Ricciardi, Fer- rara, Montesano, Castaldo Cobianchi, Vuotto & Fer- rara 2000). Regarding pharmacological activity, Kaido et al. (1997) reported on the uterotonic activity of aqueous root decoctions of G. perpensa. Joseph & Mafatle (1994) considered the effects of intraperitoneal administration of the aqueous extracts of rhizomes of the plant on the maintenance of pregnancy and parturition in rats, and discovered that the extract had a significant deleterious effect on foetuses. The authors postulated that the presence of celastrin or an unknown chemical in the extract was sufficient to kill the unborn rats, but was not enough to kill the adults (Joseph & Mafatle 1994). Antithrombin activ- ity has been detailed in a related species, Gunnera tinctoria (De Medeiros, Macedo, Contancia, Nguyen, Cunningham & Miles 2000). Recently, Khan, Peter, MacKenzie, Katsoulis, Gehring, Munro, Van Heer- den & Drewes (2004) isolated a major constituent, Z-venusol, along with the minor components pyro- gallol, succinic acid, lactic acid, and the trimethyl ether of ellagic acid glucoside from the aqueous extract of G. perpensa rhizomes. These authors demonstrated the stimulation of a direct contractile response of isolated rat uterus by the aqueous rhi- zome extract, which also induced continuous uter- ine contractility after removal of all additives from the organ bath (Khan et al. 2004). Interestingly, venusol did not trigger the contractile response but induced the state of continuous contractility after flushing of the organ bath (Khan et al. 2004). Because endometritis is an important economic dis- ease in stock farming, and resource-poor farmers frequently do not have access to orthodox veterinary medicine, we examined the biological activity of G. perpensa in more depth as part of an ethnoveteri- nary medicine project. MATERIALS AND METHODS Collection of plant material Gunnera perpensa L. rhizomes were collected in May 2002 from the Underberg region of KwaZulu- Natal. They were washed, air-dried and cut into small pieces of c. 1 cm3 using an electric band saw. The material was then milled in a rotomill (Macsa- lab) and passed through a 1 mm mesh. Preparation of extracts The dried powder (35.7 g) was extracted by vigor- ous shaking for 15 min with 150 ml hexane. The extract was removed by vacuum filtration through Whatman No. 1 filter paper using a Buchner funnel. The extraction process was repeated on the marc until the extract had little colour. Extraction was con- tinued on the marc in a serial manner using dichloro- methane, acetone, methanol and 20 % ethanol in water (ethanol/water). The extracts were subse- quently dried using a rotary vacuum evaporator (Büchi) at 40 °C. 130 Gunnera perpensa extracts in endometritis ´ ´ Antibacterial screening The dried plant extracts were dissolved to a known concentration. The acetone and ethanol/water extracts were dissolved in acetone and water re- spectively, while the remainder of the extracts were suspended in a small volume of dimethyl sulfoxide (DMSO) and made up to the required concentration with water. The test organisms in the investigation of antibac- terial activity included two Gram-positive bacteria, Enterococcus faecalis (ATCC 29212) and Staphylo- coccus aureus (ATCC 29213), and two Gram-neg- ative species, Pseudomonas aeruginosa (ATCC 27853) and Escherichia coli (ATCC 35219). The specific strains used are recommended for antibac- terial activity testing by the National Committee for Clinical Laboratory Standards (NCCLS 1990). These bacteria are responsible for most nosocomial dis- eases in hospitals (Sacho & Schoub 1993). The tetrazolium violet serial dilution microplate method of Eloff (1998) was used to evaluate the antibacter- ial activity of the extracts. This method allows the calculation of minimal inhibitory concentration (MIC) values for active plant extracts against bacte- rial species. Freshly inoculated bacterial cultures in Müller-Hinton (MH) broth were incubated overnight at 37 °C. The cultures were diluted (1:100) with MH broth before use in the assay. A twofold serial dilution of plant extract (100 µl) was prepared in 96-well microtitre plates, and 100 µl bacterial culture was added to each well. The presence of bacterial growth was detected by adding to each well 40 µl p-iodonitrote- trazolium violet (INT, 0.2 mg ml–1) that is reduced to a red-coloured formazan by respiring bacteria. When there was a substantial decrease in colour 1 hour after addition of the INT, the concentration in that well was taken to be the MIC of the plant extract against the particular bacterial strain. The antibiotic neomycin was included as a standard in each assay, and appropriate negative solvent con- trols were included. The direct thin-layer chromatography (TLC) bioauto- graphy assay (Hamburger & Cordell 1987) was employed to determine the number of antibacterial compounds and their Rf values in the Gunnera extracts. In this assay, bacterial cultures were incu- bated in MH broth overnight at 37 °C, centrifuged, and the resulting pellet resuspended in fresh MH broth. The bacteria were sprayed onto a thoroughly dried TLC chromatogram and incubated overnight at 37 °C. A solution of INT (2 mg ml–1) was sprayed on the plate and incubated again at 37 °C for approx- imately an hour. White spots appearing on a pink background of bacterial growth indicated the pres- ence of antibacterial compounds on the chromato- gram. Screening for toxicity The possible toxicity of the extracts was investigat- ed using the brine shrimp (Artemia salina) microw- ell cytotoxicity bioassay (Solís, Wright, Anderson, Gupta & Phillipson 1993). Brine shrimp eggs were obtained from a local pet shop and hatched in arti- ficial seawater (3.8 g NaCl per 100 ml distilled H2O). After 48 h, the phototropic nauplii (first stage larvae) were collected using a Pasteur pipette. The plant extracts were diluted to concentrations of 0.1, 1, 5 and 10 mg ml–1 with the artificial seawater. For each concentration, the plant extract solution was placed in two replicate wells of a 96-well microtitre plate. A suspension of nauplii (100 µl, containing approxi- mately 10–15 nauplii) was added to each well. The microplate was covered and incubated for 24 h at room temperature. The number of dead and live nauplii in each well was counted using a dissecting microscope. If control deaths occurred, the percent death values were corrected using Abbott’s formu- la: Corrected mortality percentage: m – M x 100 S m= mean percentage of dead larvae in treated tubes M= mean percentage of dead larvae in controls S = mean percentage of living organisms in controls Podophyllotoxin (Sigma) at a concentration of 5 µg ml–1 was used as a positive control and solvent blanks were included in each assay. RESULTS AND DISCUSSION Antibacterial activity The MIC values for the different bacterial species varied from 2.6–12.5 mg ml–1 (Table 1). The MIC value was higher than 12.5 mg ml–1 for all the extracts on P. aeruginosa and for the hexane extract on E. coli. McGaw et al. (2000) reported that ethanol and water extracts of G. perpensa displayed antibacter- ial activity against S. aureus, with MIC values of 3.13 and 0.78 mg ml–1, respectively. These extracts 131 L.J. McGAW et al. were prepared using separate aliquots of plant material, in contrast to the present investigation where the extracts were prepared serially with the same dried, ground plant material. The first extract- ing solvent used was hexane followed by the other solvents with increasing polarity on the same plant material. This may partially explain the absence of activity in the 20 % ethanol extract as the preceding solvents possibly removed some of the antibacterial compounds. The extract prepared using acetone had the highest antibacterial activity against the Gram-positive bacteria. Gram-positive species are generally more susceptible than Gram-negative species to plant extracts (Vlietinck, Van Hoof, Totté, Lasure, Van den Berghe, Rwangabo & Mvukiyu- mwami 1995), and this was also true in our experi- ments. According to the MIC level of 8 mg ml–1 to denote antibacterial activity of plant extracts sug- gested by Fabry, Okemo & Ansorg (1998) most extracts were inactive or had borderline antibacteri- al activity. The magnitude of the antibacterial activity depends not only on the MIC value but also on the total quan- tity of the specific extract obtained from the plant. The total activity can be calculated from the quanti- ty in mg extracted from 1 g divided by the MIC in mg ml–1 (Eloff 2000). The resultant value reflects the volume in ml to which the antibacterial com- pound can be diluted and still inhibit the growth of 132 Gunnera perpensa extracts in endometritis FIG. 1 Gunnera perpensa extracts separated on a TLC plate A: solvent system BEA (benzene:ethyl acetate:ammonia = 18:2:0.2) and sprayed with E. coli. B: solvent system CEF (chlo- roform:ethyl acetate:formic acid = 10:8:2) and sprayed with S. aureus. Extracts: H = hexane, D = dichloromethane, Ac = acetone, M = methanol, EW = ethanol/water (20 %) H D Ac M EW H D Ac M EW A B TABLE 1 Average MIC values for G. perpensa extracts (serial extraction) Escherichia coli Enterococcus Pseudomonas Staphylococcus faecalis aeruginosa aureus Extracting solvent mg extracted MICa Total MIC Total MIC Total MIC Total from 1 g mg ml–1 activity mg ml–1 activity mg ml–1 activity mg ml–1 activity ml g–1 ml g–1 ml g–1 ml g–1 Hexane 1.46 –b – 12.5 0.12 – – 6.25 0.23 Dichloromethane 3.78 12.5 0.30 5.21 0.73 – – 6.25 0.60 Acetone 69.38 12.5 5.55 4.17 16.64 – – 2.61 26.58 Methanol 54.63 6.25 8.74 3.13 17.45 – – 3.13 17.45 Ethanol/water 21.50 – – – – – – – – (20 %) Neomycin (x 10–3) 1.56 6.25 25 0.78 a MIC is taken as last concentration of plant extract to completely inhibit bacterial growth (= MLC, minimum lethal concentration) b MIC > 12.5 mg ml–1 the test organism. The highest total activity value of 26.6 ml g–1 for the acetone extract on S. aureus compares poorly with values of 182 found for Com- bretum erythrophyllum extracts on S. aureus and E. coli (Eloff 1999) and of values higher than 258 ml g–1 for Cassine peragua var. peragua (Eloff 2000). The bioautography plates are depicted in Fig. 1. The serial extraction led to some fractionation of the antibacterial compounds in G. perpensa (Fig. 1). The presence of antibacterial compounds on the bioautogram with activity against E. coli and S. aureus supports the results obtained in the micro- titre plate assay, with the hexane, dichloromethane and acetone extracts showing antibacterial activity. One compound with major activity (dichloromethane extract: Rf = 0.28) and at least three compounds with minor activity (dichloromethane extract: Rf = 0.65; hexane extract: Rf = 0.31, Rf = 0.48) against E. coli are present. The growth of S. aureus was inhibited strongly by at least four compounds (dichloro- methane extract: Rf = 0.32, Rf = 0.46, Rf = 0.63, Rf = 0.77). In all cases the active compounds were rel- atively non-polar. It has been shown that G. densi- flora and G. manicata contain caffeic acid, ellagic acid, ellagitannins and quercetin (Doyle & Scogin 1988), all of which are known to have antibacterial activity. If a chemotaxonomic relationship exists between G. perpensa and these two related spe- cies, it is possible for similar compounds to be responsible for the antibacterial activity of G. per- pensa. Brine shrimp assay The bioassay against brine shrimp larvae (nauplii) has been employed to test for the presence of toxic substances, and also as a means of facilitating the isolation of biologically active substances (Mac- Laughlin 1991, cited by Solís et al. 1993). This assay can be used to evaluate plants for pharmacological activity, taking into account the principle that phar- macology is merely toxicology at a lower dose (Vlie- tinck & Apers 2001). All of the Gunnera extracts studied were toxic at a concentration of 10 mg ml–1 (Table 2). At 1 and 0.1 mg ml–1, the hexane and dichloromethane extracts had no effect on the nau- plii. This indicates that highly non-polar compounds from G. perpensa rhizomes are not toxic to brine shrimp nauplii. The acetone extract appeared to be the most toxic, as at a concentration of 1 mg ml–1, no nauplii survived. In relation to results obtained for other plant extracts, the G. perpensa extracts are not regarded as being highly toxic. For instance, Wanyoike, Chhabra, Lang’at-Thoruwa & Omar (2004) reported toxic effects against brine shrimp larvae by a selection of Kenyan medicinal plant extracts at concentrations of less than 0.03 mg ml–1. Further tests should be performed to evaluate the potential mammalian toxicity of G. perpensa extracts, since toxicity against brine shrimp larvae is merely a preliminary indication of the likely pres- ence of toxic compounds in an extract. Neverthe- less, the toxic level for brine shrimp larvae is prob- ably considerably higher than the therapeutic dose used on animals. CONCLUSIONS Gunnera perpensa contains several compounds with antibacterial activity against E. coli and S. aureus. Based on the MIC values and total activity, it appears unlikely that the purported efficacy of G. perpensa extracts could be due solely to an anti- bacterial effect. The total antibacterial activity of the extracts was substantially lower than values found for many other plants. If one considers that aque- ous extracts are used by subsistence level farmers and that the bioactive compounds according to the bioautography results were all relatively non-polar compounds this also indicates that efficacy can probably not be ascribed to antibacterial effects of the plant extract. On the other hand McGaw et al. (2000) found that water extracts had a higher activ- ity than ethanol extracts and thus in this study it may be that first extracting plant material with non- 133 L.J. McGAW et al. TABLE 2 Percentage mortality of Gunnera perpensa extracts against Artemia salina (brine shrimp) larvae Percentage mortality at various concentrations of extract Extract 0.1 mg ml–1 1 mg ml–1 5 mg ml–1 10 mg ml–1 Hexane 0 0 14 24 Dichloromethane 0 0 92 100 Acetone 24 100 100 100 Methanol 12 69 100 100 Ethanol/water 17 36 100 100 polar extractants may have inhibited the solubility or activity of antibacterial compounds present in the G. perpensa rhizomes. In the serial extraction proce- dure, acetone and methanol may have already extracted the more polar active compounds in the rhizomes before the ethanol/water extract was pre- pared. The application of Gunnera perpensa in treating endometritis and related ailments may therefore largely be due to its previously reported uterotonic activity (Kaido et al. 1997), rather than its antibac- terial activity. This is supported by the brine shrimp assay results, which indicate a level of pharmaco- logical activity present in all the extracts. The mild effect against various bacteria demonstrated in this investigation may, however, contribute to the over- all efficacy of the traditional treatment. ACKNOWLEDGEMENTS The authors thank Dr T. Collins, who was instru- mental in obtaining plant material from the Under- berg region where subsistence farmers extensively use this traditional remedy. 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Livingstone. 134 Gunnera perpensa extracts in endometritis ´ ´