Topical formulation against gas gangrene using essential oils

Abstract

Gas gangrene is a bacterial infection which results in critical limb ischaemia and necrosis due to inadequate blood supply to the respective limb. The most common causative pathogens of gas gangrene are Clostridium perfringens and Clostridium septicum. The lack of new antibiotic developments with the increased resistance to the current antibiotic treatments has resulted in an urgent need for alternative treatments. Recent studies have indicated the antimicrobial effects of essential oils (EOs) on a vast number of pathogens, however, antimicrobial screening and toxicity studies of EOs, individually or in combination against Clostridial strains are lacking. The aim of this research was to determine the in vitro antibacterial activity of EOs, individually and in combination, against both C. perfringens and C. septicum. A topical hydrogel drug delivery system was developed for the EOs displaying the lowest minimum inhibitory concentration. A total of 56 individual EOs and 119 EO combinations were tested against the two Clostridial strains. Of the individual EOs tested, 18 and 26 EOs proved to have noteworthy inhibitory activity (MIC ≤1.00 mg/ml) against C. perfringens and C. septicum, respectively. Santalum austrocaledonicum displayed the lowest inhibitory value against both strains (MIC 0.02 mg/ml). Coriandrum sativum and Cymbopogon martinii inhibited both strains at a MIC value of 0.06 mg/ml. Five essential oils proved to be bactericidal at a noteworthy value (MBC ≤1.00 mg/ml) against both Clostridial strains. From the 119 combinations tested, 105 and 100 combinations had a noteworthy inhibition against C. perfringens and C. septicum, respectively. Santalum austrocaledonicum in combination with C. martinii displayed the lowest inhibition against both pathogens namely, C. perfringens (MIC 0.02 mg/ml) and C. septicum (MIC 0.01 mg/ml). Nine combinations have showed synergy (FIC ≤ 0.50) against both strains. Santalum austrocaledonicum and C. martinii were formulated into topical formulations individually and in combination. A Pluronic F127 and crosslinked sodium alginate hydrogel was Page | 3 developed with the EOs entrapped into micelles and within the crosslinked sodium alginate matrix. The four hydrogel formulations (three loaded and one unloaded gel) were characterised analysing the rheology; relative to the physical appearance of the gel, spreadability, EO release, degradation, antibacterial efficacy and toxicity. Rheological data indicated that the hydrogels remained in a gel state at body temperature of extremities (32 °C). This data corresponds to the physical properties and spreadability of the gels. The EO release profiles for the hydrogels were carried out in simulated wound fluid. The hydrogels formulated with S. austrocaledonicum and the combined essential oils (S. austrocaledonicum and C. martinii) respectively, both displayed an initial burst release for 12 hrs, followed by a zero-order release up to 24 hrs. The C. martinii loaded hydrogel displayed very little EO release for the first 12 hrs, thereafter a constant increase in EO release up until 60 hrs, whereupon it reached a zero-order release until 84 hrs. All four (three loaded and one unloaded gel) hydrogels displayed a high degradation rate. The hydrogels each containing essential oil S. austrocaledonicum and the combination of the oils respectively, both degraded completely within 48 hrs. The C. martinii loaded hydrogel degraded completely in 72 hrs. The unloaded hydrogel showed a 96% degradation at 72 hrs. The in vitro antibacterial efficacy of the EO loaded hydrogel was tested using the MIC microdilution assay at 32 °C. All loaded hydrogels displayed noteworthy inhibition (MIC ≤1.00 mg/ml) against both Clostridial pathogens, except for the C. martinii hydrogel against C. septicum. Hydrogels containing the two individual EOs both inhibited C. perfringens at a lower concentration in comparison to the neat EOs. The unloaded hydrogel was used as the negative control to ensure the antibacterial effect was purely due to the EO. Toxicity was determined using the brine shrimp lethality assay. Biological toxicity is considered where % mortality is greater than 50%. All four gels proved to be non-toxic after 24 and 48 hrs testing with the highest % mortality being 15%. The outcomes achieved in this study confirms proof of concept by showing the potential of selected essential oils to be used as antibacterial aids against pathogens associated with gas gangrenous infections, and that there is an urgent need for new antibacterial agents. The results show that essential oils can also successfully be entrapped and used in pharmaceutical formulations whilst displaying favourable properties for extended, effective and safe release of the bioactive oil.