View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector South African Journal of Botany 72 (2006) 637 – 641 www.elsevier.com/locate/sajb Short communication .In vitro 5-lipoxygenase inhibition and anti-oxidant activity of .Eriocephalus L. (Asteraceae) species E.W. Njenga a , A.M. Viljoen b,⁎ a Department of Pharmacy and Pharmacology, Faculty of Health Sciences, University of the Witwatersrand, 7 York Road, Parktown 2193, South Africa b School of Pharmacy, Tshwane University of Technology, Private Bag X680, Pretoria 0001, South Africa Received 27 October 2005; accepted 13 March 2006 Abstract The genus .Eriocephalus (Asteraceae) is endemic to South Africa where some of the species are used traditionally to treat dermal infections, gastro-intestinal disorders, and upper respiratory tract infections. .In vitro screening for the presence of anti-oxidants was carried out on acetone leaf extracts of 22 species (80 samples) collected from wild populations using the 2,2, diphenyl-1-picryhydrazyl (DPPH) radical scavenging method. The extracts showed moderate activity with the IC50 values ranging between 21.5 μg ml− 1 (.E. punctulatus) and 79 μg ml− 1. The hydrodistilled essential oils were also tested but did not show activity at the starting concentration of 100 μg ml− 1. Essential oils of seventeen species were screened for the presence of inhibitors of 5-lipoxygenase enzyme. The IC50 values ranged between 19 μg ml− 1 (.E. africanus) and 98.9 μg ml− 1. Variation between and within natural populations with respect to the activities tested is also documented. © 2006 SAAB. Published by Elsevier B.V. All rights reserved. The genus .Eriocephalus L. (Asteraceae) is an integral part of healing rites for various ethnic groups in South Africa and Namibia. Leaf infusions of .E. africanus were used as diuretics and diaphoretics and in the treatment of gastro-intestinal complications and gynaecological conditions. The infusions were also used in treating inflammation and other dermal complications (Watt and Breyer-Brandwijk, 1962; Van Wyk et al., 1997; Dyson, 1998; Van Wyk and Gericke, 2000). .Eriocephalus tenuifolius was used by the Griqua as a substitute for ‘buchu’ and may be due to presence of compounds with diuretic effects while .E. karooicus was also used locally as ‘wild dagga’ (Müller et al., 2001). .Eriocephalus africanus is used as a rosemary substitute for culinary flavouring purposes (Dyson, 1998). Industrially, the oils from .E. punctulatus (“Cape chamomile”) and .E. africanus (“Cape snowbush”) have a wide Abbreviations: 5-LOX, 5-lipoxygenase enzyme; DMSO, dimethylsulfoxide; DPPH, 2,2-diphenyl-1-picrylhydrazyl; NDGA, nordihydroguaiaretic acid; SANBI, South African National Biodiversity Institute; NBRI, National Botanical Research Institute; DPP, Department of Pharmacy and Pharmacology (WITS). ⁎ Corresponding author. E-mail address: viljoenam@tut.ac.za (A.M. Viljoen). application in perfumes, skin care preparations and as blend oils in beauty care products. The aromatherapeutic properties of the former oil include being an analgesic, anti-allergic, antidepressant, antiseptic, and anti-inflammatory and as a diuretic among its numerous applications (Mierendorff et al., 2003; Njenga, 2005). Plant products, whether volatile or non-volatile, are valuable sources of novel bioactive components useful in combating various diseases such as cancer, cell damage, inflammation, viral infection, allergic responses as well as in the provision of primary healthcare in most developing countries (Shale et al., 1999; Hostettmann, 1999; Heitzman et al., 2005). In the recent past, there has been an increase in the use of plants as sources of natural anti-oxidants for the scavenging of free radicals. The latter are known to initiate a series of (oxygen robbing) chain reactions resulting in oxidative tissue damage and a wide range of degenerative diseases, such as cancer, premature ageing, diabetics and a host of cardiovascular diseases (Galati and O'Brien, 2004). The rising awareness and consumer concern on issues of food preservation and safety in uses of chemical preservatives, necessitates a search for natural anti-oxidants that could not only be used to preserve food but also in the treatment of some of the diseases and their management (Sokmen et al., 2004). 0254-6299/$ - see front matter © 2006 SAAB. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.sajb.2006.03.007 638 E.W. Njenga, A.M. Viljoen / South African Journal of Botany 72 (2006) 637–641 Table 1 Anti-oxidant activity of acetone leaf extracts of species of .Eriocephalus Species Locality Voucher DPPH IC50 specimen (μg ml− 1)⁎ .E. africanus L .E. africanus .E. africanus .E. africanus .E. africanus .E. africanus .E. africanus var .paniculatus (Cass.) M.A.N. Müller .E. africanus var .paniculatus .E. africanus var .paniculatus .E. ambiguus (DC) M.A.N. Müller .E. aromaticus C.A.Sm .E. aromaticus .E. aromaticus .E. aromaticus .E. brevifolius (DC) M.A.N. Müller .E. brevifolius .E. brevifolius .E. capitellatus DC .E. decussatus Burch .E. decussatus .E. decussatus .E. decussates .E. dinteri S. Moore .E. ericoides (L.f.) Druce subsp.. ericoides .E. ericoides subsp.. ericoides Malmesbury Melkbosstrand Citrusdal (A) Citrusdal (B) Citrusdal (C) De Rust Sutherland (A) AV 444 AV 445 AV 452 AV 453 AV 454 AV 500 AV 515 47.2 ± 7.2 46.4 ± 6.7 49.9 ± 10.0 37.4 ± 8.8 38.1 ± 4.3 41.9 ± 7.1 42.5 ± 5.4 Sutherland (B) Sutherland (C) Schakalsberge AV 515 AV 519 AV 868 49.4 ± 4.4 45.8 ± 2.5 32.9 ± 2.8 Swartberg Ladismith (A) Ladismith (B) Ladismith (C) Oudtshoorn AV 484 AV 524 AV 521 AV 520 AV 483 31.8 ± 2.0 43.6 ± 4.0 45.3 ± 4.8 42.5 ± 4.5 49.7 ± 7.2 Vergelegen Kamiesberg Swartberg Pass Sutherland (A) Sutherland (B) Sutherland (C) Kamiesberg Near Aus Windhoek (Namibia) Hohenheim (Namibia) Prince Albert Scheepersrust Prince Albert Bethulie (A) Bethulie (B) Sutherland (A) Sutherland (B) Sutherland © Kamiesberg Laingsburg (A) AV 493 AV 835 AV 497 AV 532 AV 529 AV 522 AV 836 AV 871 AV 866 47.9 ± 6.2 30.9 ± 2.0 40.5 ± 3.2 47.2 ± 7.8 42.3 ± 4.5 45.9 ± 9.0 44.1 ± 4.3 34.9 ± 2.7 45.1 ± 5.0 AV 867 43.7 ± 4.4 AV 481 AV 488 AV 494 AV 747 AV 748 AV 528 AV 535 AV 534 AV 837 AV 525 47.9 ± 2.5 48.8 ± .0 56.7 ± 1.2(01) 52.7 ± 8.5 44.8 ± 3.7 56.9 ± 2.1(01) 50.3 ± 3.6 43.8 ± 8.5 39.3 ± 3.6 45.6 ± 5.1 .E. ericoides subsp.. ericoides .E. ericoides subsp.. ericoides .E. ericoides subsp.. ericoides .E. ericoides subsp.. ericoides .E. ericoides subsp.. ericoides .E. eximius DC .E. eximius .E. eximius .E. eximius .E. grandiflorus M.A.N. Müller .E. grandiflorus .E. grandiflorus .E. klinghardtensis M.A.N. Müller .E. luederitzianus O.Hoffm .E. luederitzianus .E. merxmuelleri M.A.N. Müller .E. microphyllus DC .E. microphyllus .E. microphyllus .E. microphyllus .E. microphyllus .E. microphyllus .E. microphyllus .E. microphyllus .E. namaquensis M.A.N. Müller .E. namaquensis .E. namaquensis .E. pauperrimus Merx and Eberle Laingsburg (B) Laingsburg (C) Neiaab Mountain Windhoek (A) Windhoek (B) Buschmanberge AV 533 AV 526 AV 870 46.0 ± 5.9 42.5 ± 4.1 28.1 ± 1.8 AV 865A AV 865B AV 869 48.1 ± 5.9 45.0 ± 4.9 39.9 ± 4.5 Sutherland (A) Sutherland (B) Sutherland (C) Nieuwoudtville (A) Nieuwoudtville (B) Nieuwoudtville (C) Kamiesberg Spektakel Pass Clanwilliam (A) AV 531 AV 530 AV 536 AV 542 AV 543 AV 544 AV 794 AV 795 AV 545 43.2 ± 4.2 46.2 ± 5.0 45.35 ± 5.89 44.03 ± 3.53 41.58 ± 3.87 45.56 ± 5.17 46.96 ± 6.67 47.67 ± 4.34 45.3 ± 6.47 Clanwilliam (B) AV 546 Clanwilliam (C) AV 547 Nieuwoudtville (A) AV 539 44.37 ± 4.88 44.62 ± 6.78 46.57 ± 5.82 Table 1 (.continued) Species Locality Voucher DPPH IC50 specimen (μg ml− 1)⁎ .E. pauperrimus .E. pauperrimus .E. pinnatus O.Hoffm .E. punctulatus DC .E. punctulatus .E. punctulatus .E. punctulatus .E. punctulatus .E. punctulatus .E. punctulatus .E. punctulatus .E. purpureus Burch .E. purpureus .E. purpureus .E. purpureus .E. purpureus .E. purpureus .E. purpureus .E. purpureus .E. racemosus L .E. racemosus var.racemosus .E. racemosus var.racemosus .E. racemosus var.racemosus .E. scariosus DC .E. spinescens Burch .E. spinescens .E. spinescens Control Nieuwoudtville (B) AV 540 Nieuwoudtville (C) AV 541 Brandberg AV 864 Nieuwoudtville (1A) AV 439 Nieuwoudtville (1B) AV 441 Nieuwoudtville (2A) AV 449 Nieuwoudtville (2B) AV 450 Nieuwoudtville (2C) AV 451 Nieuwoudtville (3A) AV 548 Nieuwoudtville (3B) AV 549 Nieuwoudtville (3C) AV 550 Laingsburg (A) AV 516A Laingsburg (B) AV 516B Laingsburg (C) AV 516C Nieuwoudtville (1A) AV 440 Nieuwoudtville (2A) AV 551 Nieuwoudtville (2B) AV 552 Nieuwoudtville (2C) AV 553 Kamiesberg AV 796 Koeberg AV 446 Velddrif (A) AV 455 Velddrif (B) AV 456 Velddrif (C) AV 457 Aus–Namibia AV 872 Sutherland (A) AV 523 Sutherland (B) AV 517 Sutherland (C) AV 518 Vitamin C 50.0 ± 10.84 46.46 ± 6.15 53.04 ± 4.36 43.19 ± 3.47 65.65 ± 2.76(01) 44.97 ± 4.95 32.42 ± 2.6 21.46 ± 1.29 79.63 ± 2.02(01) 38.8 ± 2.57 37.9 ± 4.06 42.33 ± 4.33 37.56 ± 4.4 37.26 ± 3.76 36.15 ± 2.99 40.05 ± 5.27 39.54 ± 4.41 38.52 ± 3.99 41.46 ± 6.03 42.88 ± 2.39 59.2 ± 9.92 40.61 ± 3.72 58.81 ± 1.7(01) 35.39 ± 3.77 41.14 ± 2.13 45.29 ± 3.88 46.47 ± 3.20 2.9 ± 0.01 ⁎ — values are means ± SE of three replicates. (A), (B), (C) represent individual plants in the same population. IC50 values are given (μg ml− 1). Despite the extensive traditional and commercial use of this indigenous genus, scientific data confirming its biological activity is lacking. On the other hand, the presence of various classes of flavonoids noted in species of this genus (Zdero et al., 1987; Wollenweber and Mann, 1989; Bohm and Stuessy, 2001; Njenga, 2005), necessitate further analysis for the presence of new biologically active constituents such as anti-oxidants. Flavonoids are known to have a broad spectrum of biochemical activities including effects on immune and inflammatory responses and thus it is important to screen the species of the genus under study to document phyto- and ethnomedical properties. In view of all these needs and the importance of phytochemical research, the present research on the members of the genus .Eriocephalus is aimed at investigating and recording the anti-oxidant and anti-inflammatory properties of the species of .Eriocephalus and provides a scientific rationale for some of the traditional uses through bioassay .in vitro screening. Fresh plant material was collected from the wild populations during the flowering and fruiting periods from different localities in South Africa and Namibia. As the study includes the aspects of variation at specific and population levels, multiple collections were made and voucher specimens prepared (Table 1). Taxonomic verification was carried out at the South African National Biodiversity Institute (SANBI) Pretoria, Compton Herbarium (Kirstenbosch) and National Botanical Research Institute (NBRI) Windhoek. The voucher specimens 639 E.W. Njenga, A.M. Viljoen / South African Journal of Botany 72 (2006) 637–641 are deposited in the Department of Pharmacy and Pharmacology (DPP) of the University of the Witwatersrand, Johannesburg, South Africa and the duplicates of Namibian taxa are deposited in the Herbarium of the National Botanical Research Institute, Windhoek, (NBRI) Namibia. Between 0.5 and 9.4 g of air-dried plant material was ground and 30 ml of acetone added. The mixture was extracted in a water bath (37 °C) for 4 h. The extract was filtered and the solvent was evaporated and resuspended in methanol. Between 20 and 750 g of the aerial plant parts (dry material) were hydrodistilled for 4 h using a Clevenger apparatus to extract the essential oils. The radical scavenging activity of the acetone leaf extracts was determined spectrophotometrically using a modified version of 2, 2, diphenyl-1-picryhydrazyl (DPPH) method of Cuendet et al. (1997) and Mambro et al. (2003). The stock solution was made by dissolving DPPH (Fluka) in analytical grade methanol to obtain a 96.2 μM solution. Extracts (5 mg) were dissolved in 500 μl of dimethyl sulfoxide (DMSO, Saarchem) to give an initial stock of 10,000 μg ml− 1. The mixture was vortexed to dissolve the extract. 50 μl of the stock solution was diluted (1:1 dilution) with 950 μl of DMSO. Then 50 μl of this stock solution was pipetted into a 96 well micro-titre plate in triplicate following the method outlined in Lourens et al. (2004). The plates were shaken on an automated micro-titre plate reader (Labsystems Multiskan RC) for 2 min and then kept in the dark at room temperature for 30 min. The changes in colour from deep violet to light yellow were measured at 550 nm on an UV/visible light spectrophotometer (Labsystems Multiskan RC) linked to the computer equipped with GENESIS® software. The radical scavenging activity was measured as the decolourization percentage of the test sample. All determinations were done in triplicate. Ascorbic acid was used as the positive control. The IC50 which is the concentration at which there is 50% decolourization of the DPPH by the test sample determined using the Enzfitter® 1.05 version software where the decolourization (%) was determined using following formula: ðAv controls−ðAv sample DPPH −Av sampleMEOH ÞÞ  100 % Decolourisation ¼ Av controls Where Av controls = average absorbance of all DPPH control wells − average absorbance of all methanol control wells; Av sampleDPPH average absorbance of sample wells with DPPH and Av sampleMEOH = average absorbance of sample wells with methanol. 5-lipoxygenase activity of the essential oils was determined using the method as published by Evans (1987) and Baylac and Racine (2003) with linoleic acid as the substrate for the 5-lipoxygenase enzyme (Cayman). In normal biological systems, 5-lipoxygenase enzyme catalyses the oxidation of unsaturated fatty acids containing 1–4 pentadiene structures with arachidonic acid as the biological substrate converting them into conjugated dienes which result in the continuous increase in absorbance at 243 nm. Standardization was first carried out using the reference sample made up of 10 μl of DMSO, 2.95 ml of phosphate buffer (pH 6.3), pre-warmed in a water bath at 25 °C. 50 μl of linoleate solution (100 μM final concentration) was added and 12 μl enzyme and 12 μl of phosphate buffer. The production of the conjugated dienes was measured over a period of 10 min at 234 nm. Two sets of controls were run with the reference samples. For the essential oils, 0.0220 g of oil was weighed and made up to a concentration of 100 μg ml− 1 in DMSO. In a 3 ml cuvette maintained at 25 °C in a thermostat bath was added 10 μl of extract, 2.95 ml of phosphate buffer (pH) 6.3, followed by 48 μl of linoleate acid (N99%, Fluka). The mixture was shaken and 12 μl of the aliquoted enzyme (stored at − 80 °C) and 12 μl of the phosphate buffer (stored at 4 °C) were pipetted to initiate enzymatic reaction. Absorbance was measured at 234 nm over 10 min using a single beam spectrophotometer (Specord 40-analytikjena) connected to a computer with Winspect® software. Linoleic acid was enzymatically converted to conjugated dienes resulting in an increase in absorbance at 234 nm. Absorbance was plotted graphically against the different concentrations used. The slopes of the straight-line portions of the sample and the control curves were used to determine the percentage activity of the enzyme (Lourens et al., 2004). The IC50 (the concentration that gives 50% enzyme inhibition) was determined using the Enzfitter® 1.05 software. Nordihydroguaiaretic acid (NDGA) was used as the positive control. Essential oils did not show any anti-oxidant activity in the DPPH assay at the starting concentration of 100 μg ml− 1 and were not further investigated. Among the acetone extracts of the taxa tested, the strongest effect was noted in.E. punctulatus from Nieuwoudtville (2C) and .E. klinghardtensis from Namibia with an IC50 of 21.5 and 28.1 μg ml− 1 respectively (Table 1). Other taxa exhibited moderate activity with IC50 vales ranging between 30 and 50 μg ml− 1. Previous studies (Zdero et al., Table 2 Anti-inflammatory (5-lipoxygenase) activity of the hydrodistilled essential oil of selected .Eriocephalus species Species Locality 5-LOX IC50 (μg/ml) .E. africanus .E. africanus .E. africanus .E. brevifolius .E. brevifolius .E. capitellatus .E. decussatus .E. dinteri .E. ericoides subsp.. ericoides .E. ericoides subsp.. ericoides .E. eximius .E. klinghardtensis .E. luederitzianus .E. merxmuelleri .E. microphyllus .E. pauperrimus .E. pinnatus .E. punctulatus .E. punctulatus .E. purpureus .E. racemosus var .racemosus .E. scariosus Control Malmesbury Melkbosstrand Citrusdal A Oudtshoorn Kamiesberg Swartberg Pass Kamiesberg Aus–Namibia Hohenheim Namibia Scheepersrust Kamiesberg Neiaab Mountain Windhoek–Namibia Buschmanberge Kamiesberg Nieuwoudtville Brandberg–Namibia Nieuwoudtville (A) Nieuwoudtville (B) Kamiesberg Velddrif B Aus–Namibia NDGA 32.8 19.0 31.8 30.2 25.4 43.1 39.6 35.4 43.1 55.4 37.9 59.7 40.5 44.5 69.4 69.9 58.7 63.0 63.8 98.9 32.8 N100 5 ± 0.5 640 E.W. Njenga, A.M. Viljoen / South African Journal of Botany 72 (2006) 637–641 1987; Wollenweber and Mann, 1989; Bohm and Stuessy, 2001) indicate that the genus produced several classes of flavonoids. Flavonoids are among the naturally occurring plant secondary metabolites that have been reported to have broad pharmacological activity including strong anti-oxidant, diuretic and diaphoretic properties. The anti-oxidant activity noted in most of the species could be attributed to the presence of flavones and flavanones that were abundant in the leaf extracts (Njenga, 2005). Further study should be conducted to evaluate their toxicity profiles and safety indices. Variation in biological activity was also noted between populations of the same species and between individuals in the same population (Table 1). These inconsistent patterns were also noted in morphology and the terpene chemistry emphasising the immense diversity in the genus (Njenga, 2005). Preliminary data indicated that the acetone leaf extracts do not inhibit the 5-LOX enzyme at 100 μg ml− 1 and given the cost of the assay the extracts were not further investigated. The oils were selected based on the availability of the oil samples as most of the taxa yielded very little oils (note: due to chemotypic variation essential oils were distilled from individual plants). The lowest effective concentration that inhibited the enzyme was 19 μg ml− 1 of .E. africanus followed by oils obtained from .E. brevifolius 25 and 30 μg ml− 1 (Table 2). Some of the compounds identified in the essential oils of these highly active species include α-pinene, β-caryophyllene, γ-terpinene, 1,8-cineole, limonene, linalyl acetate and linalool (Njenga, 2005). It is noteworthy that Baylac and Racine (2003) demonstrated that these specific terpenoids strongly inhibit the 5-lipoxygenase enzyme. The two individuals of .E. punctulatus from Nieuwoudtville have almost similar activities, as are .E. pauperrimus and .E. microphyllus. It is surprising that the former species did not show good activity despite having relatively high contents of bisabolol derivatives (Njenga, 2005). The species that showed inhibitory activity against 5-lipoxygenase enzyme included an individual of.E. africanus from Melkbosstrand that had the most promising activity (19 μg ml− 1) among all the taxa tested. Two other species, .E. brevifolius, and .E. racemosus showed moderate activity ranging between 25.4 and 32.8 μg ml− 1. Of the 17 species tested for anti-inflammatory activity, it is clear that activity varies greatly between populations (Table 2) as observed in the three populations of .E. africanus. In traditional remedies .E. africanus and .E. punctulatus are used to treat inflammatory diseases and this has been supported by the values obtained in this assay. The results also show that there are other potentially active species of .Eriocephalus not used traditionally but have inhibited the 5-LOX enzyme e.g. .E. dinteri (IC50 35 μg ml− 1), .E. brevifolius (IC50 25 and 30 μg ml− 1),.E. eximius (IC50 37 μg ml− 1) and .E. decussatus (IC50 39 μg ml− 1). The results from this study in part support the use of some members of .Eriocephalus in the treatment of inflammatory diseases mediated by 5-lipoxygenase products (e.g. leukotrienes) in traditional remedies. There is support for the use of the members of the genus in cosmetic industries as one of the properties considered is the anti-inflammatory effect. In traditional herbal remedies, some of the species are used for their soothing effects, which make them suitable for cosmetics. The ability of the oils to inhibit the 5-lipoxygenase enzyme confers credibility to the use of the ‘Cape chamomile’ and ‘Cape snowbush’ oils in cosmetics. 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