Soulll Afncon Journal of Botany 2001 67 71· 73 Pnnted lit South AfnCB - All fights reserved

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SOUTH AFRICAN JOURNAL OF BOTANY ISSN 0254·6299

Short Communication

Isolation of the anti-bacterial vernodalin from traditionally used Vernonia colorata

KA Reid, AK Jager and J van Staden'

Research Centre for Plant Growth and Development, School of Botany and Zoology, University of Natal Pietermaritzburg,

Private Bag XOI , Scottsville 3209, South Africa

• Corresponding author, e-mail: vanstadenj@nu.ac.za

Received 5 August 2000, accepted 20 September 2000

Leaf extracts of Vernonia c%rata were screened for anti-bacterial activity using the disc-diffusion assay. Micrococcus luteus, Klebsiella pneumoniae and Staphylococcus aureus were inhibited by ethyl acetate extracts , but not by hexane and water extracts . The ethyl acetate extract was fractionated by silica vacuum chromatography and a chromatron using hexane:ethyl

The genus Vernonia belongs to the family Asteraceae. It is mainly found in tropical areas, principally Africa, The com-mon name of V. colorala (Willdoenow) Drake is the lowveld tree vernonia or lowveld bitter tree. Ibozane being its Zulu name (Hutchings el al. 1996). It is an erect shrub or small tree that grows up to five metres high , The species is wide-spread in west, central and south tropical Africa, extending southwards through Mpumalanga , Swaziland and Mozambique to northern Kwa-Zulu Nata l.

Decoctions of leaves of V. colorala are used traditionally against fevers and as expectorants and laxatives. The bark of roots and stems are used for fever and diarrhoea . Roots are used as a ton ic, to treat boils, fevers and as a cough remedy (Hutchings el ai, 1996), V. colorata is used by tradi-tional medical practitioners in Zimbabwe to treat abdominal pains, bilharziasis, rheumatism, fever, infertility in women, weak joints, oedema and a painful uterus (Gel fand el al. 1985). Extracts of V. colorala are reported to have anti-amoebic, anthelmintic, anti-bacterial and parasympatholytic properties, but no anti -inflammatory activity (Hedberg and Staugard 1989, Kelmanson el al. 1999) ,

The known chemical constituents and biological proper-ties of V colorata include: vernonin which has been isolated from roots (patel and Rawson 1964), two sesquiterpene lac-tones with in vilro cytotoxic action (Hedberg and Staugard 1989) and vemolide, with anti-parasitic (Koshimuza el al. 1993) and anti-inflammatory (Satoda and Yoshi 1962, Benoit 1976) activities, Compounds with anli -inflammatory proper-ties including an unidentified sesquiterpene lactone and gly-cosides have been found in flowers and bark of V c%rata (Satoda and Yoshi 1962, Benoit 1976), A cardiac glycoside

acetate gradients . The presence of anti-bacterial activi-ty was confirmed by bioautography, The major anti-bac-terial compound in the ethyl acetate leaf extract was identified by NMR as vernodalin. The minimal inhibitory concentration of vernodalin against S. aureus was 100~g ml-1,

was isolated from stems, leaves and roots of V c%rata in Nigeria (Patel and Rawson 1964), Significant anti-malarial activity was observed for lipophilic extracts of V c%rata (Kraft et ai, 1999).

We report here on the screening of the leaves of Vernonia colorala for anti-bacterial activity to delermine if the use of V. c%rata leaves for bacteria related diseases can be sub-slantiated, The subsequent isolation of the major anti-bacte-rial compound present in the ethyl acetate leaf extract is out-lined. Leaves were col lected from a tree of Vernonia c% rata growing in the medicinal garden of the University of Natal Botanical Garden, Pietermaritzburg . A voucher specimen is deposited in the University of. Natal Herbarium (Kelmanson4UN) , Plant material was dried in an oven at 50' C for 72h. The dried material was then ground and stored in the dark at room temperature until further processing.

Plant extracts (hexane, ethyl acetate, water and elhanol) were ini tial ly tested for anti-baclerial activity using the disc-diffusion assay (Rasoanairo and Ratsimamanga-Urverg 1993). Extracts were tested against seven strains of bacte-ria obtained from the Microbiology Department of the University of Natal, Pietermaritzburg. The bacteria used were: Escherichia coli, Klebsiella pneumon;ae, Pseudomonas aeruginosa (Gram-negative) and Bacillus subtilis, Micrococcus luteus, StaplJylococcus aureus and Siaphylococcus epidermidis (Gram-positive), Stock cultures of bacteria were stored at 4°C on nutrient agar plates. Neomycin was used as a positive control. The neomycin was diluted 10 200~g ml-1, and 1 O~I then pipetted onto each disc.

72

For bioassay guided fractionation the bioautographic assay (Slusarenko el al. 1989) was used to determine the areas of anti-bacterial activity on a TLC plate. A TLC plate containing the plant extract was run with the solvent ratio of hexane: ethyl acetate 1:1 (v/v). An overn ight culture of S. aureus was grown in Mueller-Hinton (MH) broth, in a water bath at 35"C. The overnight cultures were used to inoculate new broth cultures (15ml). After 5-6h, the bacterial cells were centrifuged at 3000g for 10min at room temperature. The pellet was re-suspended in 2ml of the broth. The over-lay medium consisted of 15ml of Mueller-Hinton (MH) agar, supplemented with 15mg 2,3,5-triphenyltetrazolium chlo-ride. The bacterial suspension was added to the agar over-lay medium and the mixture was immediately poured over the whole TLC plate, in an even layer. The solidified overlay was placed on damp paper-roll in a metal tray, and sealed with plastic film, to ensure that the overlay remained moist during overnight incubation at 3rC.

Anti-bacteria l activity was only present in the ethyl acetate extract. Three bacterial strains were inhibited by the extract: M. luleus (0.53), S. aureus (1.40) and K. pneumoniae (0.53). Anti-bacterial activity is expressed as a ratio of the inhibition zone of the extracl (100mg mi") to Ihe inhibition zone of the reference (neomycin 200jJ9 ml-l). K pneumoniae is a Gram-negative bacterium, usually more resistant than Gram-posi-tive bacteria. Gram-negative P aeruginosa and E. coli strains were not inhibited by the ethyl acetate leaf extract. The highest inhibition was obtained against Gram-positive S. aureus. Gram-positive S. epidermid;s strains were no! inhibited by the ethyl acetate plant extract.

For the identification of the major active compou nd ground leaf material (205g) was defatted by Soxhlet with hexane (21 ). Further extraction of the defatted residue was carried out by Soxhlet with ethyl acetate (21). The ethyl acetate extract (35.3g) was taken to dryness and re-suspended in ethyl acetate (1 OOmg mi"). Part of this extract (3g) was frac-tionated on a vacuum silica gel column (Si lica gel 60; 0.0040 -0.063 mm) and eluted wi th a linear hexane: ethyl acetate gradient (10:0-0:10). Four hundred ml of solvent mixtu re was used. Each fraction was taken to dryness under vacu-um and the yield of each fraction determined. Activity was detected in the fraction collected during the elution with the hexane:ethyl acetate ratio of 160:240ml (v/v). This fraction was separated on a chromatron (Silica gel 60 PF 254 con-taining CaS04)' The active fraction was applied to a 1 mm thick chromalron plate and eluted wi th a gradienl of hexa-ne:ethyl acetate (10:0; 9:1; 8:2; 1:1) (v/v). One hundred ml of each solvent mixture were used. Overall , twelve fractions were collected from the ch romatron. A bioautographic assay showed the presence of anti-bacterial activity in the fraction that eluted from the chromatron with hexane:elhyl acetate 1:1 (v/v).

The isolated biologically active compound was analysed by NMR in a solution of methanol. The 13C_NMR spectrum of the sample dissolved in CD30D was recorded at 200 MHz using a Kratos MS 80RF double-focusing magnetic sector instrument at 70eV. TMS was used as an intern al standard. The 13C- NMR data for the compound with anti-bacterial activity was: 13C- NMR (50 MHZ, CH3): 6 41.0 (C-9) , 42.9 (C-10) , 47 .5 (C-5), 51.8 (C-7), 62.0 (C-19), 70.0 (C-

o

c ,

Reid, Jager and van Staden

/0 OC - C (CH,) - CH, OH

H C

° - ---i'" o Figure 1: Structure of vernoda lin, the major anti-bacterial com-pound isolated from Vernonia colorata leaves.

8) , 720 (C-14), 79 .8 (C-6), 117.4 (C-2), 121 .5 (C-13), 126.8 (C-18), 133.0 (C-4) , 135.8 (C-11), 135.8 (C-15) , 135.8 (C-17), 141.8 (C-1), 166.2 (C-16), 166.4 (C-3), 171.0 (C-12). From analyses of the NMR spectrum and comparison to published NMR-data (AI Magboul el al. 1997) it was identi-fied as vernodal in (Figure 1). This is the first identification of vernodalin in V colorata .

Minimal inhibitory concentration (MIC) for the isolated compound was determined using a serial dilution technique (Eloff 1998). An overnight culture of S. aureus was grown in MH broth , in a water bath at 35"C. The following morn-ing , 1 OO~I of the compound solution was added to the first well of a 96 well microtiter plate. The compound was serial-ly diluted with water. The overnight culture was diluted 1/100 with steri le MH broth, and 1 OO~I was added to each wel l. The microplate was covered with parafilrn and incubated at 37"C overnight. On the fina l day, 40~1 of O.2mg ml ' p-iodonitrotetrazolium violet was added to each well and the microplate was covered and incubated al 37"C for 30min . A MIC of 1 OO~g ml-1 was determined for vernodalin against S. aureus.

Vernodalin was quoted in Zdero el al. (1991) to have been isolated and identified from V colorata, but no structural data was given , and the data referred to were for vernolide (Toubiana and Gaudemer 1967) . Ho and Toubiana (1969) and Pascard (1970) referred only to the compound verno-lide. Harborne and Williams (1977) mentioned the presence of vernolide and hydroxyvernolide in V. colorala, and referred to the presence of vernodal in only in V. amygdalina. Vernodalin is known to have anti-tumor, an ti-fungal and anti-bacteria l activity. Vernolepin and vernodalin, at a concentra-tion of 1 O~g mr 1, showed anti-bacterial activity similar to that of ampicill in and neomycin at a 400~g ml-1 concentra-tion (AI Magboul el al. 1988). Vernodalin showed fu ngicidal activity at a concentration of 10~g ml-1 against Aspergillus niger and a fungistatic activi ty against Candida albicans (AI Magboul el a/. 1988). Our resulls Ihere fore substantiates the use of V colorata for bacteria re lated diseases .

Acknowledgments - We thank the Department of Microbiology, University of Natal for providing bacterial strains. the Chemistry Department for recording the NMR spectra and Prof S.E. Drewes for his help in interpreting the NMR data. Financial support was received from the National Research Foundation. Pretoria and the University of Natal Research Fund.

South Afncan Journal of Bolany 2001. 67 71 ·73

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