Antimicrobial activity of Xanthoxylum davyi and Ximenia caffra

Abstract

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.