Viljoen et(Special al. J. Essent. Oil Res., 18, 124-128 Edition 2006) Essential Oil Composition and In Vitro Biological Activities of Seven Namibian Species of Eriocephalus L. (Asteraceae) Alvaro M. Viljoen* School of Pharmacy, Tshwane University of Technology, Private Bag X680, Pretoria, 0001, South Africa Elizabeth W. Njenga and Sandy F. van Vuuren Department of Pharmacy and Pharmacology, 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 Farmaco, Universita Degli Studi di Torino, Via P. Giuria 9, 10125 Torino, Italy Abstract The essential oil composition of seven Namibian Eriocephalus species (E. dinteri, E. ericoides subsp. ericoides, E. klinghardtensis, E. luederitzianus, E. merxmuelleri, E. pinnatus, E. scariosus) were determined by GC and GC/MS. The oils of E. ericoides subsp. ericoides (sample 1), E. merxmuelleri and E. scariosus were found to be 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 accumulated high levels of α-pinene, β-pinene, p-cymene and γ-terpinene. Eriocephalus luederitzianus oil contained α-longipinene (10.3%) and β-caryophyllene (13.3%). The morphologically anomalous E. pinnatus was very different in oil composition when compared to the other taxa and is characterized by isoamyl 2-methylbutyrate (7.9%) and isoamyl valerate (6.5%). Antimicrobial disc diffusion assays and minimum inhibitory concentrations (MIC) were performed on all seven species. Good antifungal activity was noted for 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 = 35 µg/ml). Key Word Index Eriocephalus dinteri, Eriocephalus ericoides subsp. ericoides, Eriocephalus klinghardtensis, Eriocephalus luederitzianus, Eriocephalus merxmuelleri, Eriocephalus pinnatus, Eriocephalus scariosus, Asteraceae, Namibia, essential oil composition, chemotaxonomy, α-pinene, β-pinene, 1,8-cineole, santolina alcohol, linalool, chrysanthenone, camphor, α-longipinene, β-caryophyllene, antimicrobial activity, anti-inflammatory activity. 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, of which 11 occur in Namibia. Seven of these are endemic 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, dermal 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 1041-2905/06/0003-000X$14.00/0­—© 2006 Allured Publishing Corp. 124/Journal of Essential Oil Research Vol. 18 (2006) Eriocephalus Table I. Collection data and 5-lipoxygenase inhibitory activity for Namibian species of Eriocephalus Species Locality Voucher E. dinteri S. Moore E. ericoides (L.F.) Druce subsp ericoides E. ericoides (L.F.) Druce subsp ericoides E. klinghardtensis M.A.N. Müller E. luederitzianus O.Hoffm. E. merxmuelleri M.A.N. Müller E. pinnatus O. Hoffm E. scariosus DC nordihydroguaiaretic acid (NDGA) Near Aus AV 871 Windhoek district AV 866 Farm Hohenheim AV 867 Neiaab Mountain AV 870 12 km East of Windhoek AV 865 Buschmanberge AV 869 Brandberg AV 864 Near Aus AV 872 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, University 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: 6 ms). Data processing was by Hyper Chrom software (Version 2.3) (Thermo Electron Rodano, Italy). The GC/MS analyses of the oils were carried out on an Agilent 5973n GC-MS system provided with a 6890 GC unit (Agilent, Little Falls, DE, USA). Injection volume: 1 µL 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, 25 m, 0.25 mm, 0.25 µm film thickness) (Mega, Legnano (Milan), Italy). The temperature program was as follows: from 50°C (1 min) to 220°C (5 min) at 3°C/min 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 mL/min 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 mL/min, 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 of their 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 subtilis (ATCC 6051), Staphylococcus aureus (ATCC 25923), Klebsiella pneumoniae Vol. 18 (2006) Anti-inflammatory activity IC50 (µg/mL) 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 neoformans (ATCC 90112) 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 of 100 mL of agar 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 µL 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 µg per 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. Determination of minimum inhibitory concentration (MIC): 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 mg/ 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 mg/mL stock solution) for bacterial strains and Amphotericin B (0.01 mg/mL stock solution) for the yeast strains. Culture growth was visualized by transferring 40 µL of 0.2 mg/mL 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-inflammatory assay: Possible inhibition of 5-lipoxygenase activity was determined following 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 µL of each oil dissolved in Dimethyl Sulfoxide (DMSO) and Tween 20, A 0.1M potassium phosphate buffer (pH 6.3, 2.95 mL) was prepared with analytical grade reagents and 100 uM linoleic acid (≥99%). The reaction was initiated with the addition of 100 U isolated 5-lipoxygenase Journal of Essential Oil Research/125 Viljoen et al. Table II. Percentage chemical composition, yields and retention index (RI) of the oils for seven Namibian species of Eriocephalus RI Compound oil yield (%) dint 0.2 eric 1 0.2 eric 2 0.2 kling 0.2 lued 0.1 merx 0.2 pin 0.1 scar 0.4 901 921 922 928 939 939 939 963 966 970 984 988 992 996 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 santolina triene artemisia triene α-thujene α-pinene α-fenchene α-fenchene + camphene camphene sabinene β-pinene 2,6-dimethyl, 3, 5-heptadien-2-ol, myrcene yomogi alcohol α-phellandrene isoamyl isobutyrate α-terpinene p-cymene 1,8-cineole limonene santolina alcohol γ-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 α-terpineol trans-piperitol cis-piperitol methyl thymol piperitone linalyl acetate trans-chrysanthenyl acetate bornyl acetate α-longipinene neryl acetate geranyl acetate α-copaene β-caryophyllene α-humulene bicyclogermacrene δ-cadinene spathulenol caryophyllene oxide α-cadinol or Τ-muurolol β-eudesmol Total 0.2 0.9 2.8 0.3 2.5 2.3 0.4 0.7 4.2 4.4 1.3 2.2 0.5 1.9 4.5 38.4 1.2 3.4 2.9 4.6 4.1 0.7 0.3 0.8 1.6 1.8 0.7 89.8 0.7 0.3 1.0 0.6 2.0 0.6 5.4 5.6 1.2 39.0 1.5 3.3 14.3 2.1 1.1 3.2 0.9 0.2 0.5 0.2 83.9 1.7 0.3 1.2 4.3 2.8 2.1 4.8 1.6 10.4 0.7 3.6 3.0 3.2 5.0 4.3 1.1 1.7 1.8 1.0 1.4 56.2 0.5 7.9 1.2 4.4 3.8 1.0 8.9 1.4 0.6 4.3 0.4 2.2 24.4 5.4 5.6 1. 9 4.2 5.3 81.7 30.8 2.5 10.3 1.5 6.7 2.1 3.6 10.3 13.3 1.3 1.3 1.7 2.9 88.4 2.0 1.7 0.5 1.7 6.1 0.7 0.6 3.5 17.4 0.7 1.3 2.2 5.2 14.0 0.6 1.3 2.9 1.4 0.8 0.5 2.8 1.2 1.4 2.9 1.6 75.2 1.1 0.5 7.2 0.6 2.3 0.6 4.5 5.1 0.4 0.8 1.1 7.9 6.5 1.8 3.8 2.0 1.6 0.4 0.7 1.5 4.1 1.6 0.6 56.8 1.7 1.1 0.9 1.9 0.3 1.1 0.9 1.0 4.2 24.1 14.8 1.4 1.7 17.2 5.8 4.3 4.1 0.3 0.2 < 0.1 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. luederitzianus; merx = E. merxmuelleri; pin = E. pinnatus; scr = E. scariosus 126/Journal of Essential Oil Research Vol. 18 (2006) Eriocephalus 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 (IC50) was determined using Enzfitter version 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: β-pinene, p-cymene, 1,8-cineole, γ-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). Eriocephalus klinghardtensis 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. Eriocephalus merxmuelleri, E. dinteri 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 within 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 Cryptococcus neoformans (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 mg/mL to > 32 mg/mL for the oils. The lowest MIC was noted for E. merxmuelleri (2 mg/mL) against Staphylococcus aureus. Moderate to low activity was noted against Cryptococcus neoformans and Candida albicans. 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 of an 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- Table III. The antimicrobial activities of the essential oils of Namibian species of Eriocephalus. Activities are determined by disc diffusion assay (DD) measured in mm from disc edge and minimum inhibitory concentrations (MIC) in mg/mL Antimicrobial Activity Species name E. dinteri E. ericoides subsp. ericoides (AV866) E. ericoides subsp. ericoides (AV867) E. klinghardtensis E. luederitzianus E. merxmuelleri E. pinnatus E. scariosus Conventional antimicrobial control C. neoformans C. albicans DD MIC DD MIC 6.6 3.2 7.0 6.2 2.8 6.0 3.8 3.5 3.5a 32 16 8 32 * 16 16 8 1x10-3c 1.5 2.0 2.5 2.0 1.5 1.5 1.5 1.5 8.0 a 32 16 16 32 * 16 16 >32 1x10-3c B. cereus DD MIC 3.6 16 4.0 8 3.0 8 2.8 8 2.1 * 3.5 8 5.0 8 1.3 12 8.5 b 6x10-4d B. subtilis DD MIC 1.2 8 1.5 8 1.0 8 1.2 8 1.2 * 2.0 12 <1.0 * 1.0 8 6.0 b 6x10-4d S. aureus K. pneumoniae E. coli DD MIC DD MIC DD MIC 3.2 4 2.0 4 2.1 4 2.6 4 3.8 * 1.5 2 2.5 8 1.0 4 5.0 b 1x10-3d 1.5 8 1.0 16 1.5 8 2.4 8 <1.0 * 1.5 8 <1.0 * 1.5 8 2.0 b 1.3x10-3d 1.0 32 R * R * 1.0 32 R * R * R * 1.0 8 5.0 b 3x10-3d controls = aNystatin, bNeomycin, cAmphotericin B, dCiprofloxacin; 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 burn, 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 µg/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. 6. 7. 8. 9. 10. 11. 12. Acknowledgments The National Research Foundation (NRF), Medical Faculty Research Endowment Fund and the Third World Organization for Women in Science (TWOWS) are hereby acknowledged for the financial support for this study. 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