South African Journal of Botany 2002, 68: 220–222
Printed in South Africa — All rights reserved
Antibacterial activity and isolation of active compounds from fruit of the
traditional African medicinal tree Kigelia africana
OM Grace1, ME Light1, KL Lindsey1, DA Mulholland2, J van Staden1* and AK Jäger1
1 Research Centre for Plant Growth and Development, School of Botany and Zoology, University of Natal Pietermaritzburg,
Private Bag X01, Scottsville 3209, South Africa
2
Natural Products Research Group, Department of Chemistry, University of Natal Durban, Durban 4041, South Africa
* Corresponding author, e-mail: vanstadenj@nu.ac.za
Received 25 January 2001, accepted in revised form 13 June 2001
The fruits of Kigelia africana (Lam.) Benth. are a popular
source of traditional medicine throughout Africa. The
stembark has been widely analysed for pharmacologi-
cal activity, yet knowledge of the fruits is limited,
despite more extensive use in traditional remedies.
Crude extracts of stembark and fruits were prepared
with distilled water, ethanol or ethyl acetate. In the
microtitre plate bioassay, stembark and fruit extracts
showed similar antibacterial activity against Gram-neg-
Kigelia africana (Lam.) Benth. (Bignoniaceae) is a distinctive
tree native to the African continent. The indehiscent,
sausage-shaped fruits, and less commonly the pale bark of
roots and stems, are used in traditional medicines through-
out its pan-continental distribution (Retief and Herman
1997). The many uses of K. africana in therapy of physical
and magico-religious or spiritual complaints indicate that this
tree is a valuable and popular source of traditional medicine.
The diversity of ailments for which K. africana fruits and
stembark are administered in traditional African medicine
includes many microbial infections. K. africana is used in
treatment of primary and secondary infections, and as a dis-
infectant (SEPASAL 2001). Ripe or unripe fruits are dried
and powdered, and applied directly, or in topical prepara-
tions, to dermal complaints such as venereal diseases,
ulcers, acne and septic sores (Immelman et al. 1973, Burkill
1985, Pooley 1993, Hutchings et al. 1996, Van Wyk et al.
1997). Other ailments treated with medicines containing K.
africana fruits, such as complaints of the digestive and gen-
ito-urinary systems, may also be the result of bacterial infec-
tions (SEPASAL 2001).
Previous investigations of K. africana have established
antimicrobial activity of the stembark against Candida albi-
cans, Pseudomonas aeruginosa, Bacillus subtilis,
Escherichia coli and Staphylococcus aureus (Akunyili et al.
1991, Kwo and Craker 1996). Binutu et al. (1996) reported
that extracts of K. africana roots and fruits showed antibac-
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SOUTH AFRICAN JOURNAL
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Short Communication
ative and Gram-positive bacteria. A mixture of three
fatty acids exhibiting antibacterial effects was isolated
from the ethyl acetate extract of the fruits using bioas-
say-guided fractionation. Palmitic acid, already known
to possess antibacterial activity, was the major com-
pound in this mixture. These results confirm antibacter-
ial activity of K. africana fruits and stembark, and sup-
port the traditional use of the plant in therapy of bacter-
ial infections.
terial activity against Staphylococcus aureus, Bacillus sub-
tilis, and antifungal effects against Aspergillus niger,
Aspergillus flavus, Candida albicans and Pullularia pullularis.
Anti-cancer potential has been indicated by cytotoxicity of
root and bark material in the brine shrimp bioassay against
Artemia salina (Khan 1998, Khan and Mlungwana 1999).
Houghton et al. (1994) reported significant inhibitory activity
of stembark extracts against four melanoma cell lines and a
renal carcinoma cell line, and slight activity by fruit extracts.
Azuine et al. (1997) reported inhibitory effects of fruit
extracts on induced tumours and inflammation in mice.
In our study, antibacterial bioassays in which fruit and
stembark extracts were tested represented common infec-
tious agents against which traditional healers may use K.
africana. These were the Gram-positive Staphylococcus
aureus and Bacillus subtilis, and the Gram-negative
Escherichia coli and Klebsiella pneumoniae. The microtitre
plate bioassay (Eloff 1998) was used to determine bacterici-
dal effects of K. africana extracts against test organisms. An
antibacterial compound was isolated from the fruits using
bioassay-guided fractionation.
Stembark and mature fruits were collected from two local-
ities in South Africa: the National Botanical Institute Gardens
at Kirstenbosch, Cape Town, and the Department of
Agriculture Research Station at Makathini, KwaZulu-Natal.
Material was oven dried at 50°C, milled, sealed in polythene
bags, and stored in darkness at room temperature. Crude
South African Journal of Botany 2002, 68: 220–222
extracts were prepared in a ratio of 1g milled plant materi-
al:10ml solvent with distilled water, ethanol or ethyl acetate.
For the microtitre plate bioassay, aqueous extracts were
resuspended to a concentration of 50mg ml-1 in distilled
water. Ethanol and ethyl acetate extracts were resuspended
to the same concentration in acetone, to allow homoge-
neous dispersion of the sample in water. Suspension cul-
tures of test bacteria were initiated from stock cultures main-
tained at 4°C on Mueller-Hinton (MH) agar (Biolab).
Neomycin (Sigma) (100μg ml-1) was serially diluted 50%
with 100μl sterile distilled water as a positive control for each
test bacterium. Extract- and bacteria-free negative controls
were also included for each bacterium. To determine anti-
bacterial activity, 40μl p-iodonitrotetrazolium (INT) violet
(Sigma), dissolved in sterile distilled water to 0.2mg ml-1,
were added to each well. The microtitre plate was incubated
for 30min at 37°C, and colour changes verified against those
shown by controls.
Colourless INT is reduced to a red product in the presence
of metabolic activity. Test solutions in which no colour
change occurred thus represented bactericidal activity.
Minimum Lethal Concentrations (MLCs) were taken as the
lowest concentration of plant extract in wells in which colour
change did not occur. Bactericidal concentrations of the K.
africana stembark aqueous extract were 2.5mg ml-1 against
E. coli and S. aureus; activity was not detected against B.
subtilis and K. pneumoniae. Activity was not shown by the
two aqueous fruit extracts in the bioassay.
Of the ethanolic extracts, K. africana stembark showed
greatest activity against B. subtilis (MLC = 0.63mg ml-1).
MLCs of this extract against E. coli, K. pneumoniae and S.
aureus were 2.5mg ml-1. B. subtilis and K. pneumoniae
showed highest sensitivity to ethanolic extracts of fruits from
Kirstenbosch and KwaZulu-Natal respectively (MLC =
1.25mg ml-1). MLCs of fruit extracts were 2.5mg ml-1 against
the remaining test organisms.
For the ethyl acetate stembark extract, inhibitory activity
was greatest against E. coli and K. pneumoniae (MLC =
2.5mg ml-1). The ethyl acetate extract of Cape Province-col-
lected fruits was most active against B. subtilis (MLC =
0.313mg ml-1). This was the lowest MLC recorded in the
bioassay. Test bacteria responded uniformly (MLC = 2.5mg
ml-1) to the KwaZulu-Natal ethyl acetate fruit extract.
Several trends were noted in results yielded by the
microtitre plate bioassay, with significant relevance to the
use of K. africana in therapy of bacterial infections. Aqueous
extracts of stembark were active against both Gram-positive
and Gram-negative bacteria, while aqueous fruit extracts
showed no activity. Bark and fruit extracts prepared with
ethanol or ethyl acetate inhibited both Gram-positive and
Gram-negative bacteria.
Powdered material for use in traditional preparations is
usually extracted with water or spirit liquor. Aqueous infu-
sions, decoctions and ointments are prepared from material
macerated with cold or boiling water. Alcoholic preparations
are made from material macerated with water and diluted
with a fermented alcoholic suspension such as palm wine or
spirit liquor (El-Sayyad 1981, Akunyili et al. 1991, Van Wyk
et al. 1997). Results of the microtitre plate bioassay sug-
gested that more lipophilic fruit extracts elicit greater bacte-
221
riostatic or bactericidal effects than those prepared tradition-
ally. However, sensitivity shown by both Gram-positive and
Gram-negative bacteria to K. africana extracts does confirm
antibacterial properties, as Gram-negative bacteria are usu-
ally less sensitive to antibiotics than Gram-positive bacteria
(Rabe and Van Staden 1997).
Bioassay-guided fractionation was used to isolate anti-
bacterial principles in fruits collected at Makathini, KwaZulu
Natal. Dried and milled material (400g) was extracted twice
with one litre of distilled ethyl acetate for 48h at room tem-
perature. The extract was filtered and taken to dryness
under vacuum. The residue was separated by vacuum liquid
chromatography (VLC) over silica gel (Merck 230–400
mesh) using a gradient of hexane:ethyl acetate. Initially 100
% hexane was used; this was reduced to 85% hexane, and
further reduced to 50% hexane in 5% increments. Thereafter
it was decreased to 0% hexane (100% ethyl acetate) in 10%
increments.
Bioautography was used to assess antibacterial activity of
the fractions eluted by VLC. Two hundred and fifty micro-
grams of each fraction were loaded onto two TLC plates
(Merck Silica gel 60 F254, 0.25mm) and developed in a sol-
vent system of hexane:ethyl acetate (3:1). Air-dried plates
were viewed under UV366 and UV254 and bands marked. A
reference plate was sprayed with 0.5% p-anisaldehyde stain
and heated for 10min at 110°C. The test plate was allowed
to dry overnight prior to being sprayed with a concentrated
suspension of actively growing S. aureus cells. This was
prepared with bacterial cells from liquid culture collected by
centrifugation (Beckman L7-55 Ultracentrifuge) at 3 000g for
10min at 18°C. The resultant pellet was resuspended in ster-
ile MH Broth (Oxoid) to yield an optical density of approxi-
mately 0.8 (600nm). The inoculated test plate was incubat-
ed overnight at 37°C in 100% relative humidity. Antibacterial
activity was indicated by clear zones after spraying with a
2mg ml-1 solution of iodonitrotetrazolium (INT) chloride
(Fluka Chemie) and incubation for 2h. The most active frac-
tion was that eluted from the VLC with 65% hexane and 35%
ethyl acetate, and yielded 70mg.
This fraction was subjected to further TLC with a solvent
system of hexane:ethyl acetate (3:1). A strip, approximately
2.5cm wide, was cut from one chromatogram using a dia-
mond knife, and visualised with p-anisaldehyde stain. The
stained portion was re-evaluated alongside untreated plates,
and the active compound found to stain a dark green. This
band, corresponding to the dark green band on the stained
TLC plate, was scraped and recovered from the plates.
The sample was filtered through 0.22μm millipore filter
and taken to dryness, to yield 14.6mg. Chemical structures
of the active principles were determined from 1H NMR
(200MHz, CD3OD) and GC-MS data. A mixture of three fatty
acids was elucidated, of which palmitic acid was the major
compound and nonanoic acid and 8-heptadecenoic acid, the
minor compounds.
Palmitic acid has not yet been reported in K. africana, but
has exhibited antibacterial activity in several other plant
species (Hanna et al. 1996, Hashem and Saleh 1999).
Previous reports have attributed antimicrobial activity of K.
africana fruit extracts to caffeic acid and kigelinone, and
antimicrobial activity of stembark extracts to dihydroiso-
222
coumarins, naphthoquinones, iridoids and phenyl-
propanoids (Inoue et al. 1981, Akunyili et al. 1991, Houghton
and Akunyili 1993, Binutu et al. 1996).
Antimicrobial activity has been documented for other gen-
era in the Bignoniaceae. Leaf extracts of Tecomaria capen-
sis (Thunb.) Spach. ssp. capensis have shown activity
against Staphylococcus aureus and Escherichia coli
(Hutchings et al. 1996). Antimicrobial activity of Jacaranda
caucasa stembark has been attributed to betulinic acid;
Crecentia alata L. seeds have yielded oleic acid and niacin.
Antibacterial constituents of Tecoma stans L. include α-sis-
tosterol, caffeic acid, ferulic acid and indoles (Beckstrom-
Sternberg et al. 1994).
Within the context of traditional African medicine, our find-
ings are in agreement with the reputed potency of K.
africana stembark and fruits against common bacterial com-
plaints. Traditional African health care systems differ
between tribes, but the approach to diagnosis and therapy
is, in many respects, similar. Illness is considered as the
physical manifestation of disequilibria in the psychological or
social harmony of the patient (Hewson 1998). While bacter-
ial infections may not be recognised as such, the use of
effective plant drugs in traditional African therapy is well doc-
umented and, in the case of K. africana, supported by in vitro
antimicrobial activity and isolation of active principles report-
ed in this and previous studies.
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