title: Antibacterial properties of Moringa oleifera alone and in combination with conventional antibiotics
Creators: name: Ilanko, Pavithra
Publisher: name: Griffith University
Year: 2017
Subjects: subject: Moringa oleifera
subject: Conventional antibiotics
subject: Autoimmune diseases
subject: Antimicrobial phytochemicals
subject: Inhibitors

description: An increase in antibiotic resistance and a corresponding decrease in antimicrobial discovery has directed researchers towards alternative antimicrobial therapies. For centuries, humans have relied on plants for sources of effective antimicrobial agents. However, studies suggest that plants also possess the ability to potentiate the activity of other antimicrobials. Therefore, this study aims to determine the antimicrobial efficacy of Moringa oleifera extracts alone and in combination with various conventional antibiotics, against the bacterial triggers of autoimmune diseases. The study will also analyse the qualitative phytochemical and toxicity profile of the M. oleifera extracts, in order to understand the chemical and biological characteristics of a plant widely used in various traditional medicine practices throughout the world. The leaves, seed and pulp of M. oleifera was extracted using solvents of varying polarity. The antimicrobial efficacy (minimum inhibitory concentration) of these extracts were determined using a modified version of Kirby-Bauer disc diffusion and a micro-plate liquid dilution assay. The different types of interactions between M. oleifera extracts and conventional antibiotics were identified through a liquid dilution assay (fractional inhibitory concentration). Any synergistic combinations were further analysed to obtain an optimum concentration and a synergistic ratio. Furthermore, an Artemia franciscana nauplii lethality assay was used to determine the LC50 value of the M. oleifera extracts in order to determine its safety as a future chemotherapeutic drug. The qualitative phytochemical screenings show that generally, mid to higher polar extracts (water, methanol and ethyl acetate) were abundant in various antimicrobial phytochemicals including phenols, alkaloids, tannins, saponins and triterpenoids, with a few exceptions. These phytochemicals may have contributed to the greater levels of inhibition seen in mid-high polar extracts in comparison to lower polar extracts in the screenings. M. oleifera seed ethyl acetate extract performed exceptionally well in the screenings, with greater levels of activity than the ampicillin control, against Proteus vulgaris (12.6 ± 0.4mm), Acinetobacter baylyi (10.8 ± 0.8mm) and Pseudomonas aeruginosa (8 ± 0.8mm). Remarkably, twelve out of the fifteen extracts tested showed inhibitory activity against a strain of P. aeruginosa which has been previously shown to be resistant to a wide range of bacteria. A quantification of the antimicrobial efficacy as determined by minimum inhibitory concentration (MIC) of the plant revealed that most of the M. oleifera extracts inhibited the growth of at least four out of the six bacterial species tested. Generally, higher polarity extracts (methanol and water) inhibited more types of bacteria in comparison to the mid to lower polar extracts (chloroform, ethyl acetate and hexane). However, the most potent bacterial growth inhibitors were of lower polarity including M. oleifera seed chloroform (MIC - 357 μg/mL) and M. oleifera ethyl acetate (MIC - 500 μg/mL) against P. vulgaris. A total of 450 different combinations of M. oleifera extracts and five conventional antibiotics were tested against six species of bacteria. Most of the combinations were either additive or non-interactive, which suggests the combination are safe to use together. However, a few combinations produced antagonistic results. Generally, a combination of gentamicin with higher polar M. oleifera extracts (water and methanol) produced antagonistic effects against Proteus mirabilis, A. baylyi and Klebsiella pneumoniae. Therefore, it is recommended to avoid combinations of higher polarity M. oleifera extracts and gentamicin for therapeutic usage. A total of 27 different combinations (6%) were synergistic against the growth of four main bacterial triggers of autoimmune diseases. Eighteen different combinations of M. oleifera extracts (mainly lower polarity) with chloramphenicol, gentamicin and erythromycin were synergistic against the growth of P. vulgaris, a known trigger of rheumatoid arthritis. Similarly, lower polarity extracts were synergistic against the growth of P. aeruginosa and A. baylyi (triggers of multiple sclerosis). Lastly, M. oleifera leaf hexane was synergistic in combination with chloramphenicol against K. pneumoniae, a trigger of ankylosing spondylitis. Notably, several of the antibiotics that acted synergistically including penicillin, tetracycline and erythromycin were initially completely ineffective against the microbes tested. Essentially, this project has identified combinations of M. oleifera extracts that can potentiate the activity of several antibiotics that are prone to various mechanisms of resistance. However, several metabolomics (HPLC-MS, GCMS, NMR), enzyme and efflux pump inhibitory assays need to be conducted in future studies, in order to identify any potential resistance modifying agents. This study also shows that M. oleifera extracts are non-toxic and can significantly improve the efficacy of current antimicrobial drugs. Therefore, they are ideal candidates as inhibitors of the bacterial triggers of rheumatoid arthritis, multiple sclerosis and ankylosing spondylitis.