Resident skin flora are usually non-pathogenic and prevent colonization of
harmful microbes by competing for nutrients and stimulation of the immune
system. However, these resident microbes can enter the bloodstream of
immunocompromised individuals and cause life-threatening diseases.
Staphylococcus aureus and Candida albicans are examples of such microbes. S.
aureus is a pathogen responsible for the increased occurrence of nosocomial
and community-acquired infections. C. albicans causes a wide variety of fungal
infections especially in immunocompromised individuals. Due to the increased
use of antibiotics, resistant strains have increased in appearance over the past
years. As treatment options become limited, the need for novel antimicrobial
drugs becomes apparent. Most of the antibacterial drug classes known today
are derived from natural products. The aim of this study was to determine the
antimicrobial activity of bark extracts of Z. davyi and X. caffra against
planktonic and biofilm forms of C. albicans and S. aureus, as well as to
determine the phytochemistry and cytotoxicity of the crude extracts.
Zanthoxylum davyi and Ximenia caffra were chosen for evaluation based on
their ethnomedicinal uses. Hot water, methanol and dichloromethane extracts
were prepared for each plant. Thin-layer chromatography (TLC) was employed
to identify the possible classes of phytochemical compounds present in each
extract. High-performance liquid chromatography (HPLC) was used to
determine the chemical fingerprints of the plant extracts as well as to
determine the identity of phytochemicals via co-chromatography with known
standards. The disc diffusion assay was employed as a qualitative crude
screening method to identify the extracts which displayed antimicrobial
potential against two clinical and one standard strain of S. aureus and X. caffra.
The broth microdilution assay was employed to determine the minimum
inhibitory concentration (MIC) of the extracts which displayed antimicrobial
activity as determined by the disc diffusion assay. The crude extracts were
tested on biofilms of S. aureus and C. albicans using the biofilm inhibition assay with quantification by the crystal violet assay. Anti-biofilm activity was further
investigated using scanning electron microscopy (SEM) and drug interactions
were determined using the checkerboard assay which allowed for the
calculation of the fractional inhibitory concentration index. Cytotoxicity of the
crude extracts was assessed using MCF-7 human breast cancer cells and
EA.hy926 human umbilical vein cells. Effects on crude cell morphology was
visualized using phase contrast and PlasDIC microscopy.
Using TLC phenolic acids, terpenoids and sterols were detected in all extracts.
HPLC identified antimicrobial phenolic acids, flavonoids and alkaloids, with
ferulic acid, nitidine, quercetin and gallic acid in the highest concentrations.
The activity of all extracts were considered to be clinically insignificant against
planktonic S. aureus and C. albicans (>1mg/ml). All extracts displayed a
clinically significant (p < 0.05) range of concentrations which indicated antibiofilm
activity. Synergism was evident for all extracts when the two plants
were combined. From SEM analysis it was evident that the extracts caused
notable disintegration of the exopolysaccharide matrix of biofilms.
Although all the extracts displayed poor cytotoxicity, the EA.hy926 cell line was
more susceptible to the extracts than the MCF-7 cell line. This low cytotoxicity
could be ascribed to the presence of antioxidant compounds detected in all
extracts. Signs of apoptosis, such as blebbing, apoptotic bodies and nuclear
condensation, was evident in EA.hy926 cells, with visualization using phase
contrast and PlasDIC microscopy. At the highest concentrations, signs of
necrosis were observed for MCF-7 cells which include swelling and
enlargement of cells.
This study provides scientific support for the antibacterial and antifungal
activity of Z. davyi and X. caffra. It was shown that these plants could be used
as alternative antimicrobials, especially against biofilms of S. aureus and C.
albicans. To the author s knowledge, this is the first report on the antimicrobial activity of the bark extract of Ximenia caffra. Prominent antimicrobial potential
together with the low cytotoxicity supports the therapeutic potential of these
plants. Various antimicrobial phytochemicals were detected in these plant
extracts and it would appear as if antimicrobial activity is attributed to their
combined activity rather than as a result of a single compound. This study also
shows that it could be useful to combine these plants to be used as a single
antimicrobial regimen or synergistically with conventional antimicrobials. They
could also be used to increase the sensitivity of microorganisms to
conventional antimicrobials. Further research regarding isolation of the active
compounds is warranted.