Journal of Essential Oil Research ISSN: 1041-2905 (Print) 2163-8152 (Online) Journal homepage: https://www.tandfonline.com/loi/tjeo20 Essential Oil Composition and In Vitro Biological Activities of Seven Namibian Species of Eriocephalus L. (Asteraceae) Alvaro M. Viljoen, Elizabeth W. Njenga, Sandy F. van Vuuren, Carlo Bicchi, Patrizia Rubiolo & Barbara Sgorbini To cite this article: Alvaro M. Viljoen, Elizabeth W. Njenga, Sandy F. van Vuuren, Carlo Bicchi, Patrizia Rubiolo & Barbara Sgorbini (2006) Essential Oil Composition and In Vitro Biological Activities of Seven Namibian Species of Eriocephalus L. (Asteraceae), Journal of Essential Oil Research, 18:sup1, 124-128, DOI: 10.1080/10412905.2006.12067133 To link to this article: https://doi.org/10.1080/10412905.2006.12067133 Published online: 11 Jul 2019. Submit your article to this journal Article views: 2 View related articles Citing articles: 2 View citing articles Full Terms & Conditions of access and use can be found at https://www.tandfonline.com/action/journalInformation?journalCode=tjeo20 ]. Essent. Oil Res., 18, 124-128 (Special Edition 2006) Essential Oil Composition and In Vitro Biological Activities of Seven Namibian Species of Eriocephalus L. (Asteraceae) Alvaro M. Viljoen* School ofPlwnnacy, Tshwane University of Technology, Priwte Bag X680, Pretoria, 0001, South Africa Elizabeth W. Njenga and Sandy F. van Vuuren Department of Phanrwcy and Phannacology, Faculty of Health Sciences, University of the Witwatersrand, 7 York Road, Parktown, 2193, South Africa Carlo Bicchi, Patrizia Rubiolo and Barbara Sgorbini Dipartimento di Scienzia e Technologia del Fannaco, Universita Degli Studi di Torino, Via P Giuria 9, 10125 Torino, Italy Abstract The essential oil composition of seven Namibian Eriocephalus species (E. clinteri, E. ericoides subsp. ericoides, E. klinghardtensis, E.luederitzianus, E. merxmuelleri, E. pinnatus, E. scariosus) were determined by GC and GC/M S. The oils of E. ericoides subsp. ericoides (sample 1), E. merxmuelleri and E. scariosus were found to he rich in 1,8-cineole and camphor. Eriocephalus scariosus oil contained santolina alcohol (14.8%). The highest levels of camphor (38.4%) was found in E. dinteri oil. The major component of E. ericoides subsp. ericoides (sample 2) was linalool (10.4%). A chemical similarity between E. luederitzianus and E. klinghardtensis was observed which both accmnulated high p-cymene and y-terpinene. Eriocephalus luederitzianus oil contained a-longipinene levels of a-pinene, ~-pine, (13.3% ). The morphologically anomalous E. pinnatus was very different in oil composi(10.3%) and ~-caryophlen tion when compared to the other taxa and is characterized by isoamyl2-methylbutyrate (7.9%) and isoamyl valerate (fL'5%). Antimicrobial disc difTusion assays and minimum inhibitory concentrations (MIC) were perf(Jrmed on all seven species. Good antifungal activity was noted f(Jr E. ericoides subsp. ericoides. Highest activities were noted for E. merxmuelleri against the Gram-positive test organisms and generally poor activity was noted against the Gram-negative test organisms for all species. The anti-inflammatory activity of the oils were assessed using the 5-lipoxygenase (.5-LOX) enzyme and E. dinteri displayed the most promising inhibition (IC50 = 3.'5Jlglml). Key Word Index Eriocephalus dinteri, Erioceplwlus ericoides subsp. ericoides, Eriocephalus klinghanltensis, Eriocephalus luederitzianus, Eriocephalus merxmuelleri, Eriocephalus pinnatus, Eriocephalus scariosus, Asteraceae, Namibia, essential oil 1,8-cineole, santo !ina alcohol, linalool, chrysanthenone, camphor, composition, chemotaxonomy, a-pinene, ~-pine, antimicrobial activity, anti-inflammatory activity. a-longipinene, ~-caryophlen, Introduction The genus Eriocephalus L. commonly known as wild rosemary or Cape snow bush belongs to the family Asteraceae (tribe Anthemideae). The genus is characterized by presence of aromatic terpenes found in the highly dissected leaves ( 1-3). Thirty-two endemic species are reported to occur in southern Africa, ofwhich 11 occur in Namibia. Sevenoftheseareendemic to Namibia. The genus is economically important as a source of Cape chamomile oil obtained from E. punctulatus DC. and some of the species are used in traditional herbal remedies for the treatment of respiratory tract infections, gastro-intestinal disorders, dennal infections and as anti-inflammatory agents (4-8). Sesquiterpene lactones and other constituents for nine species of Eriocephalus have been reported (5). This study aims at scientifically validating traditional uses of Eriocephalus species and to report preliminary results on the oil composition of an important yet poorly studied plant group. Experimental Plant material and hydrodistillation: The aerial parts of the selected Eriocephalus species were collected during the flowering stage from various natural populations. Locality •Address for correspondence 1041-295/63X$.~ 124/Journal of Essential Oil Research 2006 Allured Publishing Corp. Vol. 1B (2006) Eriocepha/us Table 1. Collection data and 5-lipoxygenase inhibitory activity for Namibian species of Eriocepha/us Species Voucher Locality Anti-inflammatory activity IC50 (JLg/ml) E. dinteri S. Moore E. ericoides (L.F.) Druce subsp ericoides E. ericoides (L.F.) Druce subsp ericoides E. klinghardtensis M.A.N. MOiler E.!uederitzianus O.Hoffm. E. merxmuelleri M.A.N. MOiler E. pinnatus 0. Hoffm E. scariosus DC Near Aus Windhoek district Farm Hohenheim Neiaab Mountain 12 km East of Windhoek Buschmanberge Brandberg Near Aus nordihydroguaiaretic acid (NDGA) data for the studied species are given in Table I. The voucher specimens have been deposited in the Herbarium at the Department of Pharmacy and Pharmacology, {/niversity of the Witwatersrand, South Africa and the duplicates are housed in the Herbarium of the National Botanical Research Institute (NBRI), Namibia. The dried aerial plant parts were hydrodistilled in a Clevenger-type apparatus for 4 h. Essential oil analysis: GC analyses were carried out on a Thermo Electron Trace GC Ultra device provided with high frequency fast FID detector(300Hz, time constant: 6ms). Data processing was by Hyper Chrom software (Version 2.3) (Thenno Electron Rodano, Italy). The GC/MS analyses of the oils were carried out on an Agilent 5973n GC-MS system provided \Vith a 6890 GC unit (Agilent, Little Falls, DE, USA). Injection volume: 1 f.lL of each oil diluted 1:200 in cyclohexane. GC analysis conditions: Injection temperature: 250°C, mode: split, split ratio: 1:20; detector temperature: 270°C; columns: FSOT polydimethylsiloxane (OV-1, 25m, 0.25 mm, 0.25 J.lm film thickness) (Mega, Legnano (Milan), Italy). The temperature program was as follows: from 50°C (1 min) to 220°C (5 min) at 3°Chnin rate. The injector temperature was 230°C; split sampling mode, split ratio 1:10; the transfer line was 250°C, carrier gas, hydrogen at a flow rate of 1.0 mUmin in constant flow mode. GC/MS analyses were carried out on the same column under the same conditions reported for GC except that helium was used as carrier gas; flow rate: 1.0 mUmin, in constant flow mode. MS was in EI mode at 70 eV. Ion source temperature: 230°C. The components were characterized and identified by comparison oftheir mass spectra and retention indices on OV-1 with those of authentic samples or with data from literature. Percent normalization data were obtained by GC-FID. Antimicrobial activity: Two assays (disc diffusion and minimum inhibitory concentration) were employed to determine the antimicrobial activities of the oils. Disc diffusion assay: The selection of the microbial strains was carried out from a broad preliminary screening of 16 test pathogens and the seven most susceptible were selected for further disc diffusion assay (3). The disc diffusion assay was performed using five bacterial reference strains namely: Bacillus cereus (ATCC 11778), Bacillus suhtilis (ATCC 6051), Staphylococcus au reus (ATCC 25923), Klebsiella pnewnoniae Vol. 18 (2006) AV871 AV866 AV867 AV870 AV 865 AV869 AV864 AV872 35 ± 1.8 43.1 ± 3.0 59± 2.1 40.5 ± 2.5 44.5 ± 2.8 58.7±3.1 > 100 5 ± 0.5 (NCTC 9633), Escherichia coli (ATCC 8739) and two yeast strains: Cryptococcus neofimnans (ATCC 90 112) and Candida albicans (ATCC 10231). Tryptone Soya agar was prepared by dissolving 30 g of the agar in 750 mL of water and autoclaved for 15 min at 121 °C and cooled to 55°C in a water bath. A base layer oflOO mL ofagar was poured into the plate and inoculated with a top layer of 100 mL of agar containing an inoculum of approximately 1 x 106 CFU/mL. Sterilized paper discs (6 mm) were saturated with approximately 8 pL of each of the oils and loaded onto the agar plates. The plates were kept at 4°C for one hour to pre-diffuse the oil and then incubated for 24 h at 37°C for bacterial isolates. The yeasts were incubated for 48 h. Neomycin (30 pgper disc) was used as a positive control for the bacterial strains and Nystatin (100 IU per disc) as a control for the fungal strains. Activity was measured as growth inhibition zones in millimeters from the edge of the disc. Replicates were made to confirm results. Detennination ofminimwn inhihitonJ concentration (MIG): The oil yields were relatively low hence only those species with sufficient oils and with notable activity from the disc diffusion assay were included in this assay. The test was carried out using the p-iodonitrotetrazolium violet (INT) microplate method (9). The oils with a starting concentration of 128 mgl mL were transferred into the first well in the microtitre plates and serially diluted. The test cultures yielding an inoculum of approximately 1 x 106 CFU/mL were added to the wells and incubated at 37°C for 24 h for bacterial strains and 48 h for the yeast strains. The controls included were Ciprofloxacin (0.01 mglmL stock solution) for bacterial strains and Amphotericin B (0.01 mglmL stock solution) for the yeast strains. Culture growth was visualized by transferring 40 f.lL of0.2 mglmL INT to all the wells and examining them to determine the color change after 6 h for bacterial strains and 24 h for yeasts. The tests were done in triplicate. Anti-injlammatonJ assay: Possible inhibition of5-lipoxygenase activity was determined follmving published protocols (10,11). All concentrations refer to final concentrations in 3 mL cuvettes maintained at 25°C in a thermostated bath. The standard assay mixture contained 10 f.lL of each oil dissolved in Dimethyl Sulfoxide (DMSO) andTween20, A0.1M potassium phosphate buffer (pH 6.3, 2.95 mL)was prepared \vith analytical grade reagents and 100 uM linoleic acid (~9% ). The reaction was initiated \vith the addition of 100 U isolated 5-lipoxygenase Journal of Essential Oil Research/125 Viljoen et al. Table 11. Percentage chemical composition, yields and retention index (RI) of the oils for seven Namibian species of Eriocephalus Rl 901 921 922 928 939 939 939 963 966 970 984 988 992 996 1005 1008 1016 1018 1024 1047 1050 1070 1077 1083 1085 1089 1092 1110 1116 1134 1141 1141 1155 1158 1160 1166 1173 1183 1210 1215 1240 1239 1262 1336 1342 1360 1362 1398 1432 1474 1500 1544 1548 1608 1611 Compound oil yield(%) dint eric 1 eric 2 kling lued merx pin scar 0.2 0.2 0.2 0.2 0.1 0.2 0.1 0.4 0.7 0.3 1.7 1.0 0.6 2.0 0.3 santolina triene artemisia triene a-thujene a-pinene a-fenchene a-fenchene + camphene camphene sabinene 13-pinene 2,6-dimethyl, 3, 5-heptadien-2-ol, myrcene yomogi alcohol a-phellandrene isoamyl isobutyrate a-terpinene p-cymene 1,8-cineole limonene santolina alcohol y-terpinene cis-sabinene hydrate artemisia alcohol filifolone linalool isoamyl 2-methyllbutyrate isoamyl valerate chrysanthenone camphor terpinen-1-ol nerol oxide pinocamphone borneol terpinen-4-ol artemisyl acetate myrtenal a-terpineol trans-piperitol cis-piperitol methyl thymol piperitone linalyl acetate trans-chrysanthenyl acetate bornyl acetate a-longipinene neryl acetate geranyl acetate a-copaene 13-caryophyllene a-humulene bicyclogermacrene li-cadinene spathulenol caryophyllene oxide a-cadinol or T-muurolol 13-eudesmol 38.4 14.3 1.2 3.4 2.9 4.6 2.1 1.1 0.8 1.6 1.8 0.7 0.5 0.2 1.7 1.8 1.0 1.4 Total 89.8 83.9 56.2 0.2 0.9 2.8 0.3 2.5 0.6 5.4 1.7 0.5 7.9 1.2 4.4 3.8 30.8 2.5 10.3 2.0 1.1 1.7 0.5 1.7 0.5 7.2 1.1 0.9 1.9 0.3 1.1 1.2 1.5 2.3 0.4 0.7 4.2 4.4 1.3 2.2 0.5 1.9 5.6 4.3 2.8 1.2 39.0 2.1 1.5 4.8 6.1 0.7 1.0 8.9 1.4 0.6 4.3 0.4 3.3 2.1 0.6 3.5 17.4 0.7 3.6 1.3 6.7 1.6 0.9 0.6 2.3 0.6 4.5 5.1 0.4 0.8 1.1 2.2 1.0 4.2 24.1 14.8 1.4 1.7 2.2 10.4 4.5 7.9 6.5 24.4 5.2 14.0 0.7 1.8 3.8 17.2 2.0 1.6 5.8 4.3 3.6 0.6 1.3 2.9 1.4 3.0 5.4 3.2 3.2 0.4 0.7 1.5 4.1 0.8 0.5 5.0 4.1 5.6 0.9 4.3 4.1 2.8 10.3 1.6 0.7 1.1 0.3 0.3 1. 9 0.2 13.3 1.3 1.3 1.2 1.4 4.2 1.7 2.9 2.9 1.6 5.3 81.7 88.4 75.2 0.6 0.2 < 0.1 56.8 87.4 dint= E. dinteri; eric 1 = E. ericoides subsp. ericoides (AV 866); eric 2 = E. ericoides subsp. ericoides (AV 867); kling = E. klinghardtensis; lued = E. /uederitzianus; merx = E. merxmue/leri; pin =E. pinnatus; scr =E. scariosus 126/Journal of Essential Oil Research Vol. 18 (2006) Eriocephalus and E. scariosus share several compounds but this correlation in oil composition could not be supported by morphological and DNA data. The extent of variation \vithin individual species is not fully represented here as it has been noted that Eriocephalus exhibits rampant variation, both morphologically and chemically (12). A wider sampling is recommended both to fully assess and confirm relationships between species and to identify the unknown components present in the different oils. It should also be noted that the present study on the oil composition is preliminary and that an in depth investigation of the most interesting species is under way. The antimicrobial activities of the Namibian Eriocephalus oils against the test pathogens are summarized in Table III. With reference to the disc diffusion assay the oils showed activity against most of the test pathogens with highest activity noted against cry,Jtococcus neofonnans (7 mm) by the oil of E. ericoides subsp. ericoides (AV 867) and this is in agreement to other observations that essential oils are more active against yeasts than the bacteria (3,13,14). Moderate activity was noted against Bacillus cereus and Staphylococcus aureus and least activity against Escherichia coli. The oils of E. dinteri, E. ericoides subsp. ericoides and E. merxmuelleri showed the most promising activity against most of the test pathogens (Table III). Minimum inhibitory concentrations ranged from 2 mglmL to > 32 mglmL for the oils. The lowest MIC was noted for E. merxmuelleri (2 mglmL) against Staphylococcus aureus. Moderate to low activity was noted against Cnjptococcus neofonnans and Cmuli&t alhicans. In some cases there is little correlation between the inhibition diameters and MIC values and it is evident that qualitative screening methods and quantitative minimum inhibitory concentration methods are not necessarily comparable (15). The diffusion ofan essential oil in water or culture medium and the volatility of oils in the various assay systems may contribute to the incongruent results (16). Natural products have been used to regulate the process of inflammation which is a physiological body response to at- diluted with an equal volume of potassium phosphate buffer maintained at 4°C. The increase in absorbance at 234 nm was recorded for 10 min with a single beam spectrophotometer (Analytikjena Specord 40) linked to a PC by the Winaspect software. Increasing amounts of oils were added and the initial reaction rate was determined from the slope of the straight line portion of the curve. The percentage inhibition of enzyme activity was calculated by comparison with the negative control (DMSO and Tween 20). Nordihydroguaiaretic acid (NDGA) represented the positive control. Percentage enzyme activity was plotted against concentration of each oil. The concentration of each oil that caused 50% enzyme inhibition (IC 50 ) was determined using Enzfitterversion 1.05 software. In addition, single IC50 values for each oil standards identified as major compounds were determined. Results and Discussion The essential oils of the aerial parts of the seven species of Eriocephalus gave a total of 54 compounds which could be identified. Their retention indices and percentage composition are listed in Table II. Notable compounds detected in many species include: P-pinene, p-cymene, 1,8-cineole, y-terpinene, camphor, spathulenol and caryophyllene oxide. The oils of E. dinteri, E. ericoides subsp ericoides, E. merxmuelleri and E. scariosus had characteristically high contents of camphor and 1,8-cineole. The presence of camphor in E. merxmuelleri has previously been reported (5). Erioceplwlus klinglumltensis and E. luederitzianus have similar oil profiles, and it is interesting to note that in a phylogenetic reconstruction of the genus using both DNA sequence (ITS) data and chemical characters these two species were placed in the same clade (12). Both species have sericeous and opposite leaves. Eriocephalus pinnatus is one of the species in the genus that has unique autapomorphies such as yellow rays, pinnatisect leaves and absence of secondary growth in the habit. This anomaly is chemotaxonomically supported as it is the only species sampled in the greater study by Njenga (12) containing isoamyl 2-methylbutyrate and isoamyl valerate. Erioceplwlus merxmuelleri, E. dinteri Table Ill. The antimicrobial activities of the essential oils of Namibian species of Eriocephalus. Activities are determined by disc diffusion assay (DO) measured in mm from disc edge and minimum inhibitory concentrations (MIC) in mg/ml Antimicrobial Activity Species name E. dinteri B. cereus B. subtilis S.aureus DO MIC DO MIC DO MIC DO MIC DO 6.6 3.2 7.0 6.2 2.8 32 1.5 2.0 2.5 32 16 3.6 4.0 16 1.2 1.5 8 16 8 32 8 16 3.0 8 1.0 8 2.1 4 32 8 8 2.6 4 8 1.2 1.2 2.0 12 3.8 1.5 8 <1.0 2.5 1.0 1.0 4 1.5 C. neofonnans C. albicans E. ericoides subsp. ericoides (AV866) E. ericoides subsp. ericoides (AV867) E. klinghardtensis E. luederitzianus E. merxmuellerl E.pinnatus 6.0 16 2.0 1.5 1.5 16 2.8 2.1 3.5 3.8 16 1.5 16 5.0 E. scarlosus 3.5 1.5 3.5• >32 1x10-3c 1.3 Conventional antimicrobial control 8 1x10_,. 8.o• 8 12 8.5• 6x1o-•• 8 6.0• 6x10_.. K. pneumoniae E. coli MIC DO 3.2 4 1.5 8 1.0 2.0 4 1.0 16 R 1.5 8 R 8 1.0 2 2.4 <1.0 1.5 8 R R 8 <1.0 5.o• 1x10-3d MIC DO MIC 32 32 R 8 1.0 8 2.0• 1.3x10-3d 5.0 • 3x10·"' controls= •Nystatin, •Neomycin, 'Amphotericin B, "Ciprofloxacin; R = resistant; •not determined due to insufficient sample or lack of activity Vol. 18 (2006) Journal of Essential Oil Research/127 Viljoen et al. tacks by infectious organisms, or response to environmental aggressions such as sun bum, pollution, mechanical shock etc, resulting in a complex cascade of biochemical events culminating in symptoms such as redness, swelling, irritation, oedema, heat (17,18). The results in Table I show that the oils of Eriocephalus tested had the ability to inhibit 5-lipoxygenase. Eriocephalus dinteri showed the most promising inhibitory activity with an IC50 of 35 l!g/mL. It is evident from the results obtained in this study that Namibian species of Eriocephalus have potential antimicrobial and anti-inflammatory properties for treatment of dermal infections, respiratory ailments and gastro-intestinal disorders as evidenced by their activities against the relative causive pathogens and enzyme. This study provides the most recent account of the oil composition and biological properties (albeit in vitro) for some Namibian Eriocephalus species. 5. C. Zdero, F. Bohlmann and M. Muller, Sesquiterpene lactones and other constituents from Eriocephalus species. Phytochemistry, 26, 2763-2775 (1987). 6. B-E. Van Wyk, B. van Oudtshooorn and N. Gericke, Medicinal Plants of South Africa. pp 122-123, Briza, Pretoria, South Africa (1997). 7. A. Dyson, Discovering Indigenous Healing Plants of Herb and Fragrance Gardens at Kirstenbosch National Botanical Garden. pp 33-34, National Botanical Institute, Cape Town, South Africa (1998). 8. B-E. Vnn Wyk and N. Gericke, People's Plants. A Guide to Useful Plants of Southern Africa. pp 218-219, Briza, Pretoria, South Africa (2000). 9. J.N. Eloff, A sensitive and quick microplate method to determine the minimal inhibitory concentration of plant extracts for bacteria. Planta Med., 64,711-713 (1998). 10. J.C. Sircar, C.J. Shwender and E.A. Johnson, Soybean lipoxygenase inhibition by nonsteroidal anti-inflammatory drugs. Prostaglandins, 25, 393-396 (1983). 11. A. T. Evans, Actions of cannabis constituents on enzymes of arachidonate metabolism: anti-inflammatory potential. Biochem. Pharmacal., 36, 20352037 (1987). 12. E.W. Njenga, The Chemotaxonomy, Phylogeny and Biological Activity of the genus Eriocephalus L (Asteraceae). PhD thesis, Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, South Africa (2005). 13. E. Bagci and M. Digrak, Antimicrobial activity of essential oils of some Abies (fir) species from Turkey. Flav. Fragr. J., 11,251-256 (1996). 14. H.J.D. Dorman and S.G. Deans, Antimicrobial agents from plants: antibacterial activity of plant volatile oils. Appl. Microbial., 88, 308-316 (2000). 15. A.M. Janssen, J.J.C. Scheffer and A. Baerheim Svendsen, Antimicrobial activity of essential oils: A 1976 -1986 literature review. Aspects of the test methods. Planta Med., 53, 395-398 (1998). 16. A.M. Viljoen, S.F. van Vuuren, E. Ernst, M.J. Klepser, B. Demirci, K.H.C. Ba~er and B. E. VanWyk, Osmitopsis asteriscoides (Asteraceae)- The antimicrobial activity and essential oil composition ofa Cape-Dutch remedy. J. Ethnopharmacol., 88, 137-143 (2003). 17. H.P.T. Ammon, T. Mack, G.B. Singh and H. Safayhi, Inhibition ofleukotriene 8 4 formation in rat peritoneal neitrophils by an ethanolic extract of the gum resin exudates ofBoswellia serrata. Planta Med., 57, 203-207 (1991). 18. S. Baylac and P. Racine, Inhibition of 5-lipoxygenase by essential oils and other natura/fragrant extracts. Internal. J. Aromatherap., 13, 138-142 (2003). Acknowledgments The National Research Foundation (NRF), J!edical Faculty Research Endowment Fund and the Third \Vorld Organization for Women in Science (TIVOWS) are hereby acknowledgedforthefmancial support for this study. Gillian Maggs-K6lling and the staff of the National Botanical Research Institute (Namibia) are acknmvledged for the collection and identification of the plant nwterial. References 1. R.S. Adamson and T.M. Salter, Flora of the Cape Peninsula. pp 800-801, Juta, Cape Town, South Africa (1950). 2. M.A.N. MOiler, P.P.J. Herman and H.H. Kolberg, Fascicle 1: Eriocephalus and Lasiospermum. Flora of Southern Africa, 33, 1-63 (2001 ). 3. E.W. Njenga, S.F. van Vuuren and A.M. Viljoen, Antimicrobial activity of Eriocephalus L. species. S. Air. J. Bot, 71,81-87 (2005). 4. J.M. Watt and M.G. Breyer-Brandwijk, The Medicinal and Poisonous Plants of Southern and Eastern Africa. 2nd Edn., E. and S. Livingstone, London (1962). 128/Journal of Essential Oil Research Vol. 18 (2006)