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Bioactive sesquiterpene lactones from the leaves of Vernonia amygdalina

Article in Journal of Ethnopharmacology · July 2006
DOI: 10.1016/j.jep.2005.12.016 · Source: PubMed

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 Journal of Ethnopharmacology 106 (2006) 117–120

Bioactive sesquiterpene lactones from the

leaves of Vernonia amygdalina
P. Erasto, D.S. Grierson, A.J. Afolayan∗
Department of Botany, University of Fort Hare, Alice 5700, South Africa
Received 12 September 2005; received in revised form 18 November 2005; accepted 9 December 2005
Available online 3 February 2006

Abstract

Phytochemical analysis of the leaves of Vernonia amygdalina yielded two known sesquiterpene lactones: vernolide and vernodalol. The two
compounds were tested by agar dilution method against 10 bacteria strains and 5 fungi species. Both compounds exhibited a significant
bactericidal activity against five Gram positive bacteria while lacking efficacy against the Gram negative strains. In the antifungal test, while
vernolides exhibited high activity with LC 50 values of 0.2, 0.3 and 0.4mg/ml against Penicillium notatum, Aspergillus flavus, Aspergillus niger
and Mucor hiemalis, respectively, vernodalol showed moderate inhibitions against Aspergillus flavus, Penicillium notatum and Aspergillus
niger with LC50 values of 0.3, 0.4 and 0.5mg/ml, respectively. Both compounds were ineffective against Fusarium oxysporum, a microbe known
to be highly resistant to chemical agents. However, the antimicrobial results of this study correspond positively with the claimed ethnomedical
uses of the leaves of Vernonia amygdalina in the treatment of various infectious diseases. © 2006 Elsevier Ireland Ltd. All rights reserved.
Keywords: Vernonia amygdalina; Antimicrobial; Sesquiterpene lactones; Vernolide; Vernodalol; Ethnomedicine

1. Introduction sesquiterpene lactones have been identified from Vernonia

Vernonia amygdalina Del. (Asteraceae) commonly
known as bitter leaf, is a small tree growing up to 3m high.
It grows throughout the African tropics. In South Africa, it
is found in KwaZulu Natal, Mpumalanga, Eastern Cape
and Northern Provinces. Vernonia amygdalina is probably
the most used medicinal plant in the genus Vernonia. The
common and documented medicinal uses include the
treatment of schistomiasis, amoebic dysentery and
gastrointestinal problems (Huffman et al., 1996). It is also
used in the treatment of malaria, venereal diseases,
wounds, hepatitis and diabetes (Riley, 1963; Chagnon,
1984; Vlietinck et al., 1995; Akah and Ekekwe, 1995;
Hamill et al., 2000; Kambizi and Afolayan, 2001).
MembersofthegenusVernoniaaregoodsourcesofsesquiter
pene lactones. This class of compounds has been reported
to be
insectantifeedant,antifungal,cytotoxicandantitumoral(Wedg
e et al., 2000; Krishna Kumari et al., 2003). Several
amygdalina;
∗ Corresponding author. Tel.: +27 40 602 2323; fax: +27 40 602 2323. E-
mail address: Aafolayan@ufh.ac.za (A.J. Afolayan).
0378-8741/$ – see front matter © 2006 Elsevier Ireland Ltd. All rights
reserved.
doi:10.1016/j.jep.2005.12.016
these include vernolide, vernolepin, vernodalin and
hydroxyvernolide (Kupchan et al., 1969; Jisaka et al., 1993a;
Koshimizu et al., 1994). The occurrence of steroidal saponins
and flavonoids have also been reported in this plant (Rwangabo
et al., 1986; Ohigashi et al., 1991; Jisaka et al., 1992, 1993b;
Igile et al., 1994, 1995).
Very little information is available on the antimicrobial
property of compounds isolated from Vernonia amygdalina.
The work of Jisaka et al. (1993a) on the antibacterial activity of
bitter sesquiterpene lactones from this plant was limited to two
Gram positive and two Gram negative bacteria. In this paper,
we report the isolation, antifungal and more of antibacteria
activity of vernolide and vernodalol from Vernonia
amygdalina growing in South Africa. We also confirm the
identity of vernodalol as a natural product biosynthesized by
the plant rather than an artifact as suggested by a previous
worker (Jisaka et al., 1993a).
2. Materials and methods
2.1. Experimental
The 1H, 13C and DEPT 135 NMR spectra (in methanol-d4) as
118 P. Erasto et al. / Journal of Ethnopharmacology 106 (2006) 117–120
well as 2D (COSY, HMQC, HMBC) spectra were obtained on
a Bruker Avance DPX 300 (300MHz); mass spectra were
recorded on Finnigan MAT LCQ DECA instrument; melting
pointswererecordedonStuartScientific(SMP1)apparatus;vacuu
mliquidchromatography(VLC)andcolumnchromatography
(CC) experiments were achieved using silica gel 60 particle
size 0.063–0.200mm (Merck); preparative TLC was carried
out usingsilicagel60PF254+366
precoatedglassplates(Merck);analytical TLC performed on
silica gel 60 PF254 precoated alumina sheets (Merck);
visualization of compounds was done under UV lamp (254 and
266nm) and also using vanillin-sulphuric acid spray.
2.2. Plant material
The leaves of Vernonia amygdalina were collected in May
2004 from a cultivated garden in East London, South Africa.
The plant was authenticated by Prof. Afolayan and a voucher
specimen (Erasto12) deposited in the herbarium of the
University of Fort Hare.
2.3. Extraction and isolation
The air-dried leaves (500g) were pulverized and shaken in
99% ethanol at room temperature for 24h, and the extract was
concentrated under reduced pressure to afford 67g of crude
extract. It was then treated with activated charcoal to remove
chlorophyll leaving 42.6g of extract which was then subjected
to vacuum liquid chromatography using an elution gradient
from n-hexane (100%); n-hexane/CHCl3 (7:3); CHCl3 (100%);
CHCl3/EtOAc (8:2); EtOAc (100%) and finally EtOAc/MeOH
(7:3). A total of 20 fractions of 150ml each were collected.
FromthefractionselutedwithCHCl3/EtOAc(8:2)whiteneedle
like crystals formed, and were purified by recrystallizing in
CHCl3/EtOAc (9:1) to give 78mg of a compound identified as
vernolide. After spotting the remaining fractions on analytical
TLC and developing with CHCl3/acetone/MeOH (8:1:1), the
EtOAc (100%) and EtOAc/MeOH (7:3) fractions were
combined and chromatographed on silica gel column. The
elution was done using CHCl3 (100%) followed by
CHCl3/EtOAc (8:2); CHCl3/EtOAc/MeOH (7:2.5:0.5) with
increasing polarity to yield 55 fractions. Guided by analytical
TLC, fractions 30–41 were combined and subjected to
preparative TLC and developed with CHCl3/acetone/MeOH
(8:1:1) to give 243mg of a creamy feathery crystalline
compound identified as vernodalol.
2.4. Bioassay of the compounds
Adopting the method of Afolayan and Meyer (1997),
nutrient agar (NA) for bacteria and potato dextrose agar
(PDA) for fungi were prepared and autoclaved before the
compounds were
added.Totestat0.5mg/ml,5mgofacompoundwasdissolvedin
0.1mlofsolventandaddedto9.9mlofmoltennutrientmedium.
The mixture was poured into Petri dish, swirled carefully
until the agar began to set and left over night for the solvent
to evaporate. Based on the solubility of the compounds,
methanol was chosen as the solvent because it has been
reported not to inhibit the growth of bacteria at 1% level
(Dulger and Ugurlu, 2005).
2.5. Antibacterial testing
Laboratory isolates of 10 bacteria species which included
5 Gram positive and 5 Gram negative strains were obtained
from the Microbiology Department at Rhodes University.
They were; Bacillus cereus, Staphylococcus epidermidus,
Staphylococcus aureus, Micrococcus kristinae,
Streptococcus pyrogens, Escherichia coli, Salmonella pooni,
Serratia marcescens,
PseudomonasaeruginosaandKlebsiellapneumonae.Eachorga
nism
wasmaintainedonNAslants(Biolab)andwasrecoveredbycultur
ing in the nutrient broth No. 2 (Biolab) for 24h at 37◦C. Each
culture was diluted 1:100 with fresh sterile nutrient broth.
The organisms were streaked in radial patterns on agar
plates (Afolayan and Meyer, 1997). They were incubated at
30◦C and examined after 24 and 48h. A complete growth
inhibition by a specific concentration was required for it to
be declared active. The tested concentrations were 0.5, 0.25,
0.1 and 0.05mg/ml. Chloramphenicol was used as a standard
control while blank plates containing only nutrient agar and
others with nutrient agar and 1% methanol without the
compound served as blank controls. Each treatment was
replicated thrice.
2.6. Antifungal testing
Fivefungalspeciesnamely:Aspergillusflavus,Mucorhiemal
is, Fusarium oxysporum, Penicillium notatum and
Aspergillus niger were obtained from the Microbiology
Department at Rhodes University. Each culture was maintained on potato

Fig. 1. Structures of vernolide and vernodalol.

P. Erasto et al. / Journal of Ethnopharmacology 106 (2006) 117–120 119
Table 1 Table 2
Antibacterial activity of vernolide and vernodalol isolated from Vernonia 3. Results and discussion
amygdalina
Bacteriaspecies MIC(mg/ml) a Antibiotic Thephytochemicalanalysisofethanolextractfromtheleaves of
Vernolide Vernodalol Chl(0.01mg/ml) b Vernonia amygdalina afforded two bioactive sesquiterpene
lactones, vernolide and vernodalol (Fig. 1). The structures of
Gram positive
the compounds were established with the aid of NMR and MS
Bacillus cereus 0.5 0.5c +
Staphylococcus epidermidus 0.5 0.5 +
spectroscopic techniques.
Staphylococcus aureus 0.5 n.a. + Vernolide was obtained as white needle crystals with
Micrococcus kristinae 0.25 0.25 + melting point 176–177◦C, and gave an EI-MS molecular ion
Streptococcus pyrogens 0.5 0.5 + peak at m/z 362 consistent to molecular formula C 19H22O7. The
Gram negative 1
H and 13C NMR spectral data of vernolide were in good
Escherichia coli n.a. n.a.d +
Salmonella pooni 0.5 0.5 + agreement with published data (Rabe et al., 2002).
Serratia marcescens n.a. n.a. + Furthermore, vernolide has previously been reported from the
Pseudomonas aeruginosa n.a. n.a. + aerial parts of Vernonia amygdalina, Vernonia colorata and
Klebsiella pneumonae n.a. n.a. + Vernonia hindei (Zdero et al., 1991; Jisaka et al., 1993a; Rabe
et al., 2002).
a b
Minimum inhibitory concentration. Vernodalol was isolated as creamy feathery crystals with
Chloramphenicol; inhibition of bacterial growth (+). c
melting point 133–135◦C, and gave an EI-MS molecular ion
Maximum concentration of the compounds tested. d
Not active. peak at m/z 392 consistent to molecular formula C 20H24O8. The
1
H NMR spectral data of vernodalol were in agreement with
the earlier report of this compound by Asaka et al. (1977).
dextrose agar (PDA) and was resuscitated for testing by Jisaka et al. (1993a) reported vernodalol to be an artifact of
subculturing on fresh PDA for 3 days. The prepared plates vernodalin, presumed to be the product of methanol and
containing compounds at concentrations of 0.5, 0.25, 0.1 and extract solvolysis reaction during extraction process. However,
0.05mg/ml, respectively were inoculated with plugs obtained based on the fact that crude extraction in this study was done
from actively growing margin of the fungi plates and using ethanol, coupled with the fact that vernodalol was first
incubated at 25◦C for 4 days. The plates containing nystatin identified by Asaka et al. (1977) from the dried seeds of
served as standard control for each organism. Diameter of Vernonia anthelmintica it is evident that this compound is not
fungal growth was measured and expressed as means of an artifact. The quantity of vernodalol obtained from the
percentage growth inhibition of three replicates. Significant extract was also very high (243mg/500g of dried leaves) which
differences within the means of the measurements and the further suggests that vernodalol is biosynthesized by the plant
control were calculated using the LSD statistical test (Steel itself.
and Torrie, 1960). The LC50 values were calculated by Vernolide and vernodalol exhibited significant antibacterial
extrapolation. activity against all the Gram positive bacteria strains tested
(Table 1). Both compounds showed high activity against
Antifungal activity of vernolide and vernodalol isolated from Vernonia amygdalina
Compound Concentration (mg/ml) Growth inhibition (%)

AF MH FO PN AN
a a a a
Vernolide 0.5 64.8 60.3 39.5 74.9 59.7a
0.25 51.9b 15.2b 26.7b 56.9b 29.1b
 0.1 35.7c 6.1c 21.9b 43.0c 20.3c

0.05 1.8d 3.6c 12.8c 31.2d 7.3d

Methanol 2.1d 0.0d 2.1d e 0.0e

Control 0.0d 0.0d 0.0d 0.0e

e
LC50 (mg/ml) 0.3 0.4 – 0.2 0.4

Vernodalol 0.5 62.3a 11.2a 28.6a 62.5a 50.9a

0.25 46.0b 6.7b 23.9a 37.2b 29.1b

0.1 32.2c 3.3c 7.3b 19.3c 23.3c

0.05 11.4d 0.0d 2.5c 20.4c 17.9d

Methanol 2.1e 0.0d 0.0c 0.0d 0.0e

Control 0.0e 0.0d 0.0c 0.0d 0.0e

LC50 (mg/ml) 0.3 – – 0.4 0.5

Nystatin (g/ml)a <25 <25 <75 <25 <25

Values are means of percentage growth of three replicates; values within a column followed by the same superscript of the same compound are not
significantly different at P<0.05 according to the LSD test. LC50 values were calculated by extrapolation. AF, Aspergillus flavus; MH, Mucor hiemalis; FO,
Fusarium oxysporum; PN, Penicillium notatum; AN, Aspergillus niger. a Nystatin: standard antifungal drug.
120 P. Erasto et al. / Journal of Ethnopharmacology 106 (2006) 117–120
Micrococcus kristinae with the MIC value of 0.25mg/ml.
However, the two compounds lacked efficacy on the Gram
negative bacteria with the exception of Salmonella pooni
which was inhibited by both compounds at 0.5mg/ml.
Sesquiterpene lactones are known to be highly antifungal
(Wedge et al., 2000), the fact which was also demonstrated by
vernolide and vernodalol in this study (Table 2). Vernolide
exhibited higher antifungal activity against Penicillium
notatum and Aspergillus flavus with LC50 values of 0.2 and
0.3mg/ml,
respectively.Itfurtherdemonstratedasignificantactivityagainst
Aspergillus niger and Mucor hiemalis with LC50 values of
0.4mg/ml each. However, vernolide was less effective against
Fusarium oxysporum where the highest growth inhibition was
39.5%. This fungus is known to have high resistance to most
fungicides (Wedge et al., 2000). Vernodalol generally
exhibited weak fungicidal activity compared to vernolide.
However, it demonstrated a significant activity against
Aspergillus flavus, Penicillium notatum and Aspergillus niger
with the LC50 values of 0.3, 0.4 and 0.5mg/ml, respectively.
Thisstudyhasdemonstratedsomestructure–
activityrelationships in the two compounds. They both belong
to elemanolide lactone system. Both compounds have an ,
-unsaturated carbonyl system, but differ in the number of
hydroxyl groups and the presence of an epoxide ring in
vernolide. Vernodalol have two hydroxyl groups while
vernolide is mono-hydroxylated, which makes the former to be
more polar and less lipophilic than the later. Lipophilicity has
been suggested to be the factor influencing fungicidal activity
of sesquiterpene lactones and other commercial fungicidal
drugs. An increase in lipophilicity increases the toxicity of the
compounds on fungi (Beekman et al., 1997; Wedge et al.,
2000). This study has provided a scientific support for the
claimed ethnomedical uses of the leaves of Vernonia
amygdalina in the treatment of various infectious diseases.
Acknowledgements
This work was supported by the National Research
Foundation, South Africa. Technical assistance of Prof. R.R.T.
Majinda as well as S. Marape and M. Chacha of Chemistry
Department, University of Botswana is highly appreciated.
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