Antifungal Activity of Naphthoquinones and Triterpenes Isolated from the Root Bark of Euclea Natalensis ⁎ N

South African Journal of Botany 72 (2006) 579–583 www.elsevier.com/locate/sajb

Antifungal activity of naphthoquinones and triterpenes isolated from the root bark of Euclea natalensis ⁎ N. Lall , O. Weiganand, A.A. Hussein, J.J.M. Meyer

University of Pretoria, Botany Department, Pretoria 0002, South Africa Received 2 June 2005; accepted 29 March 2006

Abstract

Euclea natalensis A. DC. belonging to the family Ebenaceae, is widely used for curing bronchitis, pleurisy, chronic asthma and urinary tract infections by the Zulus, in South Africa. Four triterpenes namely lupeol, betulin, β-sitosterol, 20(29)-lupene-3β-isoferulate and two naphthoquinones shinanolone and octahydroeuclein isolated from the ethanolic extract of E. natalensis root bark were investigated for their antifungal activity against Aspergillus flavus, Aspergillus niger, Cladosporium cladosporioides,andPhytophthora sp. A. niger was significantly inhibited by β-sitosterol, 20 (29)-lupene-3β-isoferulate and shinanolone at 0.01 mg ml−1. Of all the compounds tested, only octahydroeuclein was found to be significantly effective against Phytophthora sp. at 0.1 mg ml−1. β-Sitosterol and octahydroeuclein inhibited the growth of C. cladosporioides significantly at 0.01 mg ml−1. None of the isolated compounds exhibited significant activity against A. flavus at 0.01 mg ml−1. © 2006 SAAB. Published by Elsevier B.V. All rights reserved.

Keywords: Euclea natalensis; Triterpenes; Napthoquinones; Antifungal

1. Introduction common on the East Coast of South Africa. The roots of this plant are being used for curing bronchitis, pleurisy, chronic Fungal pathogens are capable of causing serious diseases in asthma and urinary tract infections by the Zulus. The roots are humans and animals (Van Burik and Magee, 2001). Fungal also used as a dye, for skin infections caused by Mycobacterium infections are commonly seen in patients suffering from AIDS. leprae and to relieve headache and toothache by the local (Kwong-Chung and Bennett, 1992). Aspergillosis caused by inhabitants of South Africa (Palgrave and Drummond, 1977). Aspergillus species is a rare complication of AIDS and usually According to previous investigations, roots of E. natalensis seen in patients with advanced disease (Ioachim, 1989). Serious have been found to be antibacterial against Mycobacterium ailments such as Phaeohyphomycosis, pneumonia, necrotising tuberculosis and a number of Gram-positive and Gram-negative enterocolitis, are caused by fungi like Cladosporium clados- bacterial species (Lall and Meyer, 1999, 2000; Khan et al., porioides, Phytophthora sp. (Hallstrom et al., 2004; Vieira et 1978). The aim of the current study was to investigate the ac- al., 2001). Due to problems with the toxicity of existing tivity of the purified compounds from the root bark of E. antifungal agents as well as the emergence of drug-resistant natalensis against various fungal species. This is the first report strains, the use of plants can be exploited as an alternative way on the inhibitory effect of compounds isolated from Euclea to control these pathogens. Many plants contain secondary natalensis root extracts against fungal pathogens. compounds that have been found to have an inhibitory effect on harmful bacterial and fungal human pathogens (Lall and Meyer, 2. Material and methods 2000; Magama et al., 2002; Masika and Afolayan, 2002; Meyer and Afolayan, 1995). 2.1. Plant material Euclea natalensis A. DC. belonging to the family Ebena- ceae, is widely distributed in tropical, subtropical Africa and is Roots of E. natalensis were collected from Mputaland, a region in the KwaZulu–Natal province of South Africa. A ⁎ Corresponding author. voucher specimen (N.L. 22) was identified and deposited at the E-mail address: [email protected] (N. Lall). H.G.W.J. Schweickerdt Herbarium (PRU), Pretoria.

2.2. Fungal strains 0.01 mg ml− 1. Three blank plates containing PDA-agar and 2% acetone served as controls. Bifonazole (Srbolek, Belgrade) and Four fungal species, Aspergillus flavus, Aspergillus niger, metalaxyl (Sungbo Chemical Co, Korea) were used as positive C. cladosporioides, and Phytophthora sp. used in this study drug control. Once the agar had solidified, a 5 mm plug of a 7- were obtained from the Department of Microbiology and Plant day-old fungal culture was placed in the centre of the Petri Pathology, University of Pretoria. All strains were maintained dishes containing the compound-amended and acetone- on potato dextrose agar (PDA) at ±25 °C. amended PDA plates. The plates were sealed with Parafilm and placed in an incubator at 25 °C. Fungal growth was mea- 2.3. Isolation of compounds sured on two preset diametral lines after 2, 3, and 4 days of growth. Each treatment was analysed in triplicate. The results of Four triterpenoids namely lupeol, betulin, 20(29)-lupene-3β- the 4-day growth was statistically analysed using two-way isoferulate, β-sitosterol and two naphthoquinones, octahy- analysis of variance (ANOVA) and least significant differences droeuclein and shinanolone (Fig. 1a–f) were isolated and puri- (P=0.05) were determined according to Student's t test. fied from the root bark of E. natalensis, as mentioned in our previous paper (Weigenand et al., 2004). The chemical struc- 3. Results and discussion tures of the compounds were determined by spectroscopic methods (1H and 13C NMR) and comparison with authentic A. niger was significantly inhibited by β-sitosterol, 20(29)- samples. A stock solution (10 mg ml− 1) of each compound was lupene-3β-isoferulate and shinanolone at 0.01 mg ml−1. Of all the prepared in acetone and stored at 4 °C until further analysis. compounds tested, only octahydroeuclein was found to be significantly effective against Phytophthora sp. at 0.1 mg ml−1. 2.4. Antifungal bioassay β-Sitosterol and the naphthoquinone, octahydroeuclein inhibited the growth of C. cladosporioides significantly at 0.01 mg ml−1, For the antifungal assay, the required amount of compounds similar to the findings of other researchers where a novel were added to sterile PDA in 65 mm Petri dishes before naphthoquinone, ‘Eleutherinone’, was found to have strong activ- congealing to yield final concentrations of 0.1, 0.05 and ity in the direct bioautographic assay with the phytopathogenic

Fig. 1. Compounds isolated from the root bard of Euclea natalensis: (a) lupeol, (b) betulin, (c) 20(29)-Lupene-3β-isoferulate, (d) β-Sitosterol, (e) shinanolone, (f) octahydroeuclein. N. Lall et al. / South African Journal of Botany 72 (2006) 579–583 581 fungus Cladosporium sphaerospermum. In another study, 1,4- β-Sitosterol was found to have good inhibitory activity against naphthoquinone-2-yl and naphthoquinone derivatives 6–9and all the tested fungi in this study except against A. flavus.The 2,3-disubstituted 1,4-naphthoquinones were also found to be antifungal activity of β-sitosterol isolated from Dioscorea alata antifungal (Tandon et al., 2004). Bifonazole, a positive drug has been shown as inhibitor of the elongation of germ tubes of control, inhibited the growth of A. niger, A. flavus and C. Fusarium molniliforme as high as 82% at 50 mg ml−1.The cladosporioides at 15 μg/ml whereas ‘metalaxyl’ inhibited the findings of the present study are in confirmation with antifungal growth of Phytophthora sp. at 30 μg/ml. activity of β-sitosterol from Senecio lyratus in in vitro

Fig. 2. Antifungal activity of (a) lupeol, (b) betulin, (c) 20(29)-lupene-3β-isoferulate, (d) β-sitosterol, (e) shinanolone, (f) octahydroeuclein on selected fungal pathogens. ⁎Each value of a bar is a mean of three replicates. Values of the bars within each fungal species not followed by the same letter are significantly different (P=0.05) according to the Student's t-test. 582 N. Lall et al. / South African Journal of Botany 72 (2006) 579–583

Fig. 2 (continued). investigations by Kiprono et al. (2000).However,noantifungal indigenous people of South Africa. The results show a fairly high activity of β-sitosterol isolated from Artemisia sp. was correlation between antifungal activity of E. natalensis root bark observed (Tang et al., 2000). None of the isolated compounds and the traditional medicinal use by tribes of Southern Africa. exhibited significant activity against A. flavus at 0.01 mg ml− 1 (Fig. 2a–f). Acknowledgements Crude extract and semi purified fractions obtained from Eu- clea crispa have been found to have inhibitory effect on human The Carl Duisberg Foundation of Germany, NRF (South pathogenic fungi (Magama et al., 2002). The results of this work Africa) are acknowledged. Thanks are due to Mr. L. Govender suggested that the suppression of fungi growth supports the use of chairman of Herbal Associations, Durban, Mr. Eric Palmer from root bark of E. natalensis for different types of infections by Department of Chemistry for their valuable assistance. N. Lall et al. / South African Journal of Botany 72 (2006) 579–583 583

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