Journal of Ethnopharmacology 93 (2004) 207–212

Biological activity of five antibacterial flavonoids from

Combretum erythrophyllum (Combretaceae)
N.D. Martini, D.R.P. Katerere, J.N. Eloff∗
Programme for Phytomedicine, Department of Paraclinical Sciences, University of Pretoria, Pretoria 0002, South Africa
Received 10 July 2003; received in revised form 28 February 2004; accepted 28 February 2004

Available online 11 June 2004

Abstract

Preliminary studies with Combretum erythrophyllum showed antimicrobial activity against Gram-positive and Gram-negative bacteria.
Seven antibacterial flavonoids were subsequently isolated by bioassay-guided fractionation, i.e. apigenin; genkwanin; 5-hydroxy-7,4 -dime-
thoxyflavone, rhamnocitrin; kaempferol; quercetin-5,3 -dimethylether; rhamnazin. All compounds had good activity against Vibrio cholerae
and Enterococcus faecalis, with MIC values in the range of 25–50 ␮g/ml. Rhamnocitrin and quercetin-5,3 -dimethylether also inhibited
Micrococcus luteus and Shigella sonei at 25 ␮g/ml. With the exception of 5-hydroxy-7,4 -dimethoxy-flavone the flavonoids were not toxic
towards human lymphocytes. This compound is potentially toxic to human cells and exhibited the poorest antioxidant activity whereas
rhamnocitrin and rhamnazin exhibited strong antioxidant activity. Genkwanin; rhamnocitrin; quercetin-5,3 -dimethylether; rhamnazin had a
higher anti-inflammatory activity than the positive control mefenamic acid. Although these flavonoids are known, this is the first report of
biological activity with several of these compounds.
© 2004 Elsevier Ireland Ltd. All rights reserved.

Keywords: Combretum erythrophyllum; Antimicrobial; Antioxidant; Anti-inflammatory; Toxicity; Flavonoids

1. Introduction
Plants have developed an arsenal of chemicals to sur-
vive attacks by microbial invasion (Grayer and Harborne,
1994). These include both physical barriers as well as chem-
ical ones, i.e. the presence or accumulation of antimicrobial
metabolites. These metabolites are either preformed in the
plant (prohibitins) or induced after infection (phytoalexins).
Since phytoalexins can also be induced by abiotic factors
such as UV irradiation, they have been defined as ‘antibiotics
formed in plants via a metabolic sequence induced either bi-
otically or in response to chemical or environmental factors’.
When infection or damage to a plant takes place, a number
of processes are activated and some of the compounds pro-
duced become activated immediately whereas phytoalexins
are produced after two to three days. Sometimes it is dif-
ficult to determine whether the compounds are phytoalex-
ins or prohibitins especially as the same compound may be
a preformed antimicrobial substance in one species and a
phytoalexin in another. The chemical classes in which these
∗ Corresponding author. Tel.: +27 12 319 2139; fax: +27 12 319 2411.
E-mail address: jneloff@medic.up.ac.za (J.N. Eloff).
substances can be found varies greatly and many are identi-
fied as flavonoids (Cowan, 1999).
Combretum erythrophyllum is widely used in traditional
medical practice in southern Africa. It has been used for
treating abdominal pains and venereal diseases, which indi-
cates the presence of antibacterial compounds in the leaves
(Hutchings et al., 1996). The aim of this study was to iso-
late the antibacterial constituents of this species since leaf
extracts showed substantial activity against Staphylococ-
cus aureus, Enterococcus faecalis, Staphylococcus aureus
and Pseudomonas aeruginosa (Martini and Eloff, 1998;
Eloff, 1999). Bioassay-guided fractionation yielded seven
flavonoids, all but one isolated for the first time from Com-
bretaceae (Martini et al., 2004). Flavonoids are well docu-
mented for their biological effects, including antimicrobial
and cardiovascular activity, which led to the belief that a
diet rich in fruit and vegetables contributes to good health
(Williamson et al., 2000). Large gaps still exist, however,
particularly regarding their pharmacological effects and
therefore antimicrobial, antioxidant and anti-inflammatory
bioassays were performed on the isolated flavonoids to shed
more light on these phenolic metabolites.
There are currently no recommended and acceptable dos-
ing regimens for commercially available “bioflavonoids” and

0378-8741/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.jep.2004.02.030
208 N.D. Martini et al. / Journal of Ethnopharmacology 93 (2004) 207–212

few toxicological reports (Berkoff, 1998). We also attempted 5 min at 1200 rpm (400 × g) after adding 40 ml PBS and
to determine the cytotoxicity of the isolated flavonoids us- the resulting pellet (lymphocytes and monocytes) washed
ing the lactate dehydrogenase (LDH) assay. This is a pop- again with PBS. Lysis buffer (0.83% (w/v) NH4 Cl) was
ular and reliable test for cytotoxicity in immunological as added to the remaining cells to induce hypotonic lysis
well as biocompatibility studies (Allan and Rushton, 1994). of the contaminating erythrocytes and suspended cells
Several studies have been done on kaempferol and apigenin were incubated on ice for 5–6 min before centrifuging for
and they were therefore not included in the bioassays. 5 min at 1000 rpm. This procedure was repeated and the
remaining pellet resuspended in 1–5 ml PBS after which
cells were counted and adjusted to a concentration of
2. Materials and methods 107 ml−1 with PBS. All cells were kept in an ice bath prior
to use.
2.1. Plant material, extraction and isolation
2.3.2. Lactate dehydrogenase assay (LDH)
The procedure described in Martini et al. (2004) was Lactate dehydrogenase (LDH) is a cytosolic enzyme
followed for the isolation work. Briefly leaves were present within all mammalian cells. The normal plasma
dried, extracted with acetone, separated into fractions by membrane is impermeable to LDH, but damage to the cell
solvent–solvent fractionation, the chloroform fraction was membrane results in a change in the membrane permeabil-
fractionated by open column and closed column Silica gel ity and subsequent leakage of LDH into the extracellular
chromatography and collected by crystallization. fluid. In vitro release of LDH from cells provides an accu-
rate measure of cell membrane integrity and cell viability
2.2. Test organisms used (Allan and Rushton, 1994).
Neutrophils (107 cells/ml) were incubated with Hanks’
One fungus and 10 bacterial isolates were used as shown balanced salt solution (HBSS) for 5 min at 37 ◦ C in a ra-
in Table 1. Minimum inhibitory concentrations were deter- tio of 1:4, i.e. 20 ␮l cells + 80 ␮l HBSS. As a positive
mined by a serial dilution microplate assay using tetrazolium control, 20 ␮l lysophosphatidylcholine (LPC) was added to
violet as growth indicator (Eloff, 1998). one Ependorff tube and the volume adjusted to 200 ␮l with
HBSS. Two other controls were selected, one with distilled
2.3. Toxicity assay water and the other with the same ratio of DMSO to water in
which the compounds were brought to solution and 100 ␮l of
2.3.1. Cell separation each added to their respective tubes. In the other Ependorffs
Heparinized blood was taken from healthy volunteers 100 ␮l of the test compounds were added into their respec-
and diluted in a ratio of 1:2 with phosphate-buffered tive tubes to make up a final concentration of 100 ␮g/ml.
saline (PBS). The diluted blood was layered onto Ficoll The tubes were incubated for a further 10 min at 37 ◦ C af-
(Histopaque 1077, Sigma Diagnostics) in a ratio of 2:1 ter which they were centrifuged at 1000 rpm for 5 min. The
and centrifuged for 30 min at 2100 rpm (1000 × g). The supernatant (100 ␮l) of each was pipetted and transferred to
serum was discarded up to 2 mm from the top layer and a microplate into which 100 ␮l PBS was added. Into each
the mononuclear cells were aspirated and removed into well 20 ␮l of NADH (1.3 mg/ml) was added and just before
a clean 50 ml tube. The suspension was centrifuged for reading 20 ␮l pyruvate (1.1 mg/ml) was added to initiate the
reaction. The microplate was read at 340 nm at time 0, 1, 5
and 10 min and measurements were done in triplicate.
Table 1
Test organisms used in this study showing the time of incubation and
2.4. Antioxidant/anti-inflammatory activity
positive controls used for MIC determination
Organism Positive Class Incubation 2.4.1. Serum opsonization of zymosan
control period (h)
Zymosan A (Sigma Chemical Co.) was mixed with dis-
Aspergillus niger AB Fungus 24
tilled water (2.5 mg/ml), boiled for 5 min and centrifuged
Bacillus subtilis G Gram-positive 24
Escherichia coli G Gram-negative 4 at 900 rpm for 5 min until sedimented. The zymosan was
Klebsiella pneumoniae G Gram-negative 4–6 washed with 10 ml PBS and resuspended before centrifug-
Micrococcus luteus A Gram-positive 24 ing at 900 rpm for 5 min. Freshly pooled serum (10 ml) from
Pseudomonas aeruginosa G Gram-negative 4 a healthy volunteer was added to the zymosan and incubated
Salmonella typhimurium A Gram-negative 4
for 60 min at 37 ◦ C.
Shigella sonei A Gram-negative 4
Staphylococcus aureus A Gram-positive 4 After centrifugation (900 rpm, 5 min), the supernatant was
Enterococcus faecalis A Gram-positive 4 discarded and the pellet washed twice with PBS before
Vibrio cholerae C Gram-negative 12–24 finally resuspending in saline solution at a concentration
G: gentamicin; A: ampicillin; C: chloramphenicol; AB: amphotericin B. of 2.5 mg/ml and used as the serum opsonized zymosan
Mueller Hilton Broth was used as growth medium for all organisms. (SOZ).
 N.D. Martini et al. / Journal of Ethnopharmacology 93 (2004) 207–212 209

2.4.2. Preparation and chemiluminescence assay

Isolated test compounds were dissolved in dimethylsul-
foxide (DMSO) and distilled water to yield a concentration
of 200 ␮g/ml. The final concentration for each stimulant in
solution with lymphocytes was: SOZ, 279 ␮g/ml and com-
pounds, 50 ␮g/ml.
In each cuvette 100 ␮l of the cell suspension (2×106 cells/
ml), was incubated with 100 ␮l luminol (5-amino-2,3-
dihydro-1,4-phthalazinedione; Sigma; final concentration
10−4 M) and the test compounds (100 ␮l) for 30 min at
37 ◦ C. The cuvettes were inserted in the chemiluminome-
ter and the stimulant (SOZ) added just before recording.
A sample that was not stimulated with SOZ but contained
cells, luminol and RPMI to adjust the volume was included
as the blank control. Two other controls, one containing Fig. 2. Flavonols isolated from Combretum erythrophyllum. RC: rhamnoc-
RPMI instead of test compound and the other DMSO and itrin; KP: kaempferol; QM: quercetin-5,3 -dimethylether; RN: rhamnazin.
water in the same concentration used to suspend com-
pounds, were also included in the assay. Measurements were
Comparing the MIC values of the other isolated com-
done in triplicate and each sample measured for 30 min
pounds (Table 2) with initial data (Martini and Eloff, 1998;
using a luminescence assay. All buffers and reagents were
Martini, 2002), the most active antimicrobial compounds
used from the same batch to limit variations in results. The
present in the extracts were not isolated or alternatively there
peak response (peak CL) and the time for peak CL (peak
must have been a synergistic effect between different com-
time, minutes) were read from the recorder (BioORbit Oy,
pounds. The standard antibiotics used as controls had much
1991).
lower MIC values than the test compounds. In this study
no pentacyclic triterpenes were isolated and it is known that
compounds such as erythrophyllic acid occur in Combretum
3. Results erythrophyllum (Rogers, 1998). Katerere et al. (2003) could
show that imberbic acid, a pentacyclic triterpene from Com-
The structures of seven flavonoids isolated from Com- bretum imberbe, has a MIC value of 3.13 ␮g/ml towards
bretum erythrophyllum (Martini et al., 2004) are shown in Staphylococcus aureus.
Figs. 1 and 2. Unpredictable and unreliable results were obtained when
the microplate serial dilution method was applied to As-
3.1. Antibacterial activity pergillus niger. Some compounds seemed to enhance the
growth of the fungus, whereas in other wells, the same com-
Since several studies have shown both kaempferol and pound showed inhibition. Also no growth was observed in
apigenin to possess antimicrobial activity, they were there- wells with dilute test compounds. There was no apparent in-
fore not included in the bioassays (Bisignano et al., 2000; hibition at the concentrations used (100 ␮g/ml) and the size
Cai and Wu, 1996; Kurosaki and Nishi, 1983; Basile et al., and colour of the organisms in the wells were the same as
1999). those of the growth control. The growth period of 24 h may
have been too short.

Table 2
MIC values (␮g/ml) of some of the compounds isolated from Combretum
erythrophyllum leaf extracts
HM QM RN RC GK

Salmonella typhimurium >100 >100 >100 >100 >100

Klebsiella pneumonia >100 >100 >100 >100 >100
Micrococcus luteus 100 25 >100 25 50
Escherichia coli 50 50 100 50 100
Staphylococcus aureus >100 >100 >100 50 >100
Shigella sonei 50 25 >100 25 25
Vibrio cholerae 25 50 50 25 50
Pseudomonas aeruginosa 100 100 100 100 100
Enterococcus faecalis 50 50 50 50 50
Bacillus subtilis >100 >100 >100 >100 >100
Fig. 1. Flavones isolated from Combretum erythrophyllum. AP: apigenin;
Aspergillus niger >100 >100 >100 >100 >100
GK: genkwanin; HM: 5-hydroxy-7,4 -dimethoxyflavone.
210 N.D. Martini et al. / Journal of Ethnopharmacology 93 (2004) 207–212
Test compounds exhibited similar activities against indi-
vidual microorganisms, possibly due to similarities between
structures and hence structure–activity relationships. There
does not appear to be a vast difference in inhibitory activ-
ity between Gram-positive and Gram-negative organisms,
whereas the pentacyclic triterpenes did not inhibit growth of
the Gram-negative Escherichia coli at 100 ␮g/ml, the highest
level tested (Katerere et al., 2003). With some compounds
there appears to be a bacteriostatic rather than bactericidal
activity as wells previously showing inhibition grew out af-
ter prolonged incubation periods.
The best activity was seen against Vibrio cholerae
(25–50 ␮g/ml) and Enterococcus faecalis (50 ␮g/ml), which
were inhibited by all five compounds. Inhibition of Pseu-
domonas aeruginosa and Escherichia coli was between
50 and 100 ␮g/ml and all compounds, with the exception
of rhamnazin, exhibited activity against Shigella sonei
(25–50 ␮g/ml). Rhamnocitrin also inhibited the growth of
Staphylococcus aureus with an MIC value of 50 ␮g/ml,
whereas this organism proved resistant to all other com-
pounds. Good activity was also observed against Micrococ-
cus luteus by quercetin-5,3 -dimethylether and rhamnocitrin
(25 ␮g/ml) and genkwanin (50 ␮g/ml).
Antibacterial activity has been previously shown
against some periodontal pathogens by rhamnocitrin and
5-hydroxy-7,4 -dimethoxyflavone (Cai and Wu, 1996; Wang
et al., 1992). Salmonella typhimurium, Klebsiella pneumo-
nia, Bacillus subtilis and Aspergillus niger were all resistant
to all the test compounds at 100 ␮g/ml, the highest level
tested.
3.2. Toxicity assay
Yokozawa et al. (1999) suggested that the number and
position of phenolic hydroxyl groups linked to a structural
backbone largely decide the difference in activity. This is
possibly the reason why most of the compounds exhibited
similar toxicity profiles (Fig. 3). Most of the compounds
Fig. 3. The average percentage LDH released by LPC, water, water
+ DMSO and five test compounds.
have similar toxicity profiles to that of water. Since wa-
ter in large volumes can cause lysis of cells, this could
possibly explain the increase in LDH levels. DMSO/water
was used as solvent for the compounds and did not ap-
pear to influence the cells to any large extent and showed
values lower than that of water alone. Rhamnocitrin and
quercetin-5,3 -dimethylether had similar profiles to that of
the solvent showing therefore a toxicity less than that of
water, whereas genkwanin and rhamnazin were close to
that of water. This appeared to indicate that these com-
pounds were no more toxic than the indirect effect caused
by water. In some cases (bioassay was repeated in triplicate
and the average taken) 5-hydroxy-7,4 -dimethoxyflavone
had shown values very close to those obtained with
LPC and since its influence on cell lysis was greater
than that of water it can be presumed to be potentially
toxic.
3.3. Antioxidant/anti-inflammatory activity
Luminol-dependent chemiluminescence (CL) has been
recognised as a useful tool for evaluating the phagocytic ac-
tivity of granulocytes and lymphocytes. Changes in counts
of luminol-dependent CL can represent the effects of various
drugs on the functions of these cells (Ozaki et al., 1984).
Luminol is converted to an excited aminophthalate ion in
the presence of oxidising compound and this reaction emits
blue light measured by the chemiluminometer. The chemical
basis of the chemiluminescence reaction is not known in
detail but superoxide anion and the myeloperoxidase product
hypochlorite (HOCl) are necessary for generating luminol
amplified chemiluminescence (Wiik et al., 1996).
Lymphocytes were used primarily due to ease of iso-
lation and greater stability in comparison to neutrophils
whose life span is considerably shorter. A blank (cells
+ luminol + zymosan) was included as negative control and
mefenamic acid (100 mg/ml), a commercial non-steroidal
anti-inflammatory drug, as positive control. Cells were
stimulated with opsonised zymosan.
Results show, as expected, the blank control emitting the
greatest percentage of light (in millivolts) due to superoxide
production (Fig. 4). The solvent (DMSO + water) had vir-
tually no effect on the free radical production and therefore
did not interfere with activity of the test compounds. Mefe-
namic acid is a commercially available anti-inflammatory
and analgesic preparation with moderate anti-inflammatory
activity. It is expected to reduce inflammatory components
suppressing the reaction. All compounds were compared to
this positive control.
Genkwanin showed greater suppression of the curve than
mefenamic acid and was assayed as an in vitro antioxidant by
its ability to inhibit tert-butyl hydroperoxide initiated chemi-
luminescence of mouse liver homogenates (IC50 > 1 ␮M)
(Fraga et al., 1987). It is a potential water-soluble protector
against lipid peroxidation and other free radical-mediated
cell injury. Quercetin-5,3 -dimethylether had a similar
 N.D. Martini et al. / Journal of Ethnopharmacology 93 (2004) 207–212
Fig. 4. Free radical production (mV) produced by lymphocytes after
stimulation of cells incubated with water and DMSO (W + D) and various
test compounds. C is the cells alone incubated in RPMI and zymosan
was used as stimulant throughout.
activity to genkwanin. This is the first report of such activity
by quercetin-5,3 -dimethylether.
Both rhamnocitrin and rhamnazin showed strong antiox-
idant effects confirming the results of Manez et al. (1999)
and Yun et al. (2000). Rhamnocitrin was found to exert
anti-inflammatory action and exhibit effectivity against 12-
O-tetradecanoylphorbol-13-acetate-induced ear oedema in
mice, although lacking activity against arachidonic acid-
induced oedema (Manez et al., 1999). Rhamnazin signif-
icantly inhibited lipid peroxidation in rat liver microsome
(Yun et al., 2000).
Although free radical production was inhibited by 5-
hydroxy-7,4 -dimethoxyflavone, it had the weakest activ-
ity and therefore does not have significant antioxidant
activity. Looking at the structure–activity relationship, a
possible reason for the poor activity of 5-hydroxy-7,4 -
dimethoxyflavone could be the methoxy group positioned
at C-4 . All the other compounds have a hydroxy group
at C-4 , which does not appear to be influenced by a C-5
methoxy group (QM and RN). Changing the positioning of
the OH and OCH3 groups on C5 and C7 also appears to
have no effect on the antioxidant activity seeing that GK
and QM have very similar activities.
Kaempferol, which is already known to have a remarkably
high free radical-scavenging activity, was not tested.
4. Discussion and conclusion
Phenolic substances generally have significant antimicro-
bial activity and it is assumed that their function in tissues
where they accumulate might be to provide chemical barri-
ers to invading microorganisms. It has been postulated that
the methylated, lipophilic flavonoids are especially suit-
able as protection against microorganisms because of the
ease with which they penetrate bacterial cells (Williamson
et al., 2000). Many possess antifungal properties, especially
the nonpolar polymethylated flavones and the prenylated
isoflavones as well as various chalcones and flavanones.
211
The pterocarpan phytoalexins, which have antimicrobial
activities and are characteristic of members of Legumi-
nosae occur in soybean, alfalfa and chickpea (Ibrahim,
2000). Flavonoids have been reported to be more active
against Gram-negative bacteria, but other studies indicate
that Gram-positive bacteria are selectively inhibited by
flavonoids and isoflavonoids derived from plants. Basile
et al. (1999) suggest that the pattern of selectivity towards
Gram-positive bacteria is not restricted to compounds from
plants but is a general observation amongst most antibiotics.
In this study the flavonoids isolated inhibited the growth
of Gram-positive and Gram-negative organisms at similar
levels and MIC values for Enterococcus faecalis, Vibrio
cholerae, Shigella sonei, Escherichia coli and Micrococcus
luteus ranged between 25 and 100 ␮g/ml. Structure–activity
relationships do not appear to play a role here as all seven
compounds exhibited similar MICs against the organisms
tested and a wider range of organisms as well as chemi-
cal derivatives will have to be included in such a bioassay
for a more accurate interpretation. Although the levels of
antibacterial activity of these compounds do not justify
the use of Combretum erythrophyllum in the treatment of
infections caused by the organisms tested, leaf extracts or
leaf sap could possibly be used in the purification of water
contaminated by Vibrio cholerae. This is an avenue yet to
be explored and could be a low-tech solution to cholera in
rural areas if the sap or extracts have a high therapeutic
index.
It is likely that the anti-inflammatory activity found in
Combretum extracts (Eloff et al., 2001) could be ascribed to
flavonoids.
Research also supports the antioxidant properties of
flavonoids and their ability to reduce the risk of developing
cardiovascular disease, possibly due to inhibition of oxi-
dation of low-density lipoprotein (LDL). They also inhibit
inflammation by decreasing the release of inflammatory me-
diators and by stabilizing cell membranes (Berkoff, 1998).
Many flavonoids exhibit remarkably high radical-scavenging
activity indicating perhaps that they could reduce potential
neoplastic and inflammatory effects attributed to free radical
formation (Sawa et al., 1999). The most powerful antioxi-
dants are to be found among the flavonoids and several are
now acknowledged as having 20 times the potency of Vita-
min C and 50 times the potency of Vitamin E. Flavonoids
are essential for processing Vitamin C and are needed to
maintain capillary walls as well as protect against infection.
Rutin is an example of a flavonoid that helps increase cap-
illary strength and ensures fast healing. A deficiency can
result in a tendency to bruise easily (Berkoff, 1998).
Of the five flavonoids tested, all exhibited antioxidant and
potential anti-inflammatory activity. Most also had higher
activity than mefenamic acid, a commercially available
non-steroidal anti-inflammatory drug. If a hydroxy group
at C-4 is replaced by a methoxyl group in 5-hydroxy-
7,4 -dimethoxyflavone it could be the reason for the poor
activity of this flavone.
212 N.D. Martini et al. / Journal of Ethnopharmacology 93 (2004) 207–212
Although flavonoids are consumed in great quanti-
ties from the foods we eat there are no reported toxi-
city levels for these compounds. Many flavonoids have
however been found to be toxic to cancer cells and are
currently being investigated for this use (Lopez-Lazaro
et al., 2000). Flavonoids in this study proved no more
toxic to lymphocytes than water except for 5-hydroxy-7,4 -
dimethoxyflavone, which appeared in most cases to have
values closer to LPC indicating toxicity. As previously men-
tioned, this compound also exhibited poor antioxidant and
anti-inflammatory activity, although antibacterial activity
was comparable to that of the other compounds.
The results provide a clear rationale for the ethnomedici-
nal use of Combretum erythrophyllum leaves in treating in-
fections.
Acknowledgements
This research was supported by the National Research
Foundation, The Technology and Human Resources for In-
dustry Programme and Biomox Pharmaceuticals. Mrs. A.
Lombard (Microbiology, U.P.) supplied the microorganisms
and Dr. A. Theron (Immunology, U.P.) assisted with the tox-
icity assays.
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