Comparison of in vitro antimicrobial activity of Cannabis extracts and modified isoniazid derivatives against drug resistant Candida and Mycobacteria species

Abstract

Microbial resistance to their respective treatment methods of microorganisms has become a major problem in the public health sector. Both Candida and Mycobacterium species have been noted as multi-drug resistant species which result in treatment difficulties. Various methods of treatment have been undertaken for both fungal and mycobacterial infections. Resistance is due to overexposure and overuse of drugs as well as shortened treatment courses which evade the drug’s mechanism of action. Medicinal plants are viewed as an alternative form of treatment which can be evaluated for the treatment and management of infections caused by these re-sistant microorganisms. Another form of treatment that has been recently explored is the re-purposing of drugs. This study was set to focus on the antioxidant, cytotoxic and antimicrobial activity of Cannabis indica/sativa extracts and modified isoniazid derivatives. Six Cannabis indica/sativa extracts derived from vegetative leaves and mature inflorescence parts were evaluated; these plant parts were extracted by means of the Soxhlet extraction method using three solvents namely: n-hex-ane, ethyl acetate and ultrapure water. Modified isoniazid derivatives were received from an ongoing study. However, they also form an important part of the study. The 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging assay was used to evaluate the antioxidant scav-enging potential. These were measured using the inhibitory concentration at which 50% (IC50) of the radicals have been scavenged of both extracts and isoniazid derivatives. Of the tested agents, the ethyl acetate extract of the mature inflorescence was the best radical inhibitor with an IC50 = 34.8μg/mL and among isoniazid derivatives, N’-((4-aminophenyl) phenyl) meth-ylene) isonicotinohydrazide· (salicylic acid) (IBS 010) was the strongest IC50 = 105.4μg/mL. The cytotoxicity assay was performed to determine which concentration of the Cannabis in-dica/sativa extracts and modified isoniazid derivatives would kill 50% of the murine macro-phages (RAW 264.7) and African green monkey kidney (Vero) cells (LC50). The plant extracts and modified isoniazid derivatives were evaluated in triplicates, the vegetative leaves of n-hexane extract showed the best activity against the Vero cells with an LC50 = 2 404.6μg/mL and mature inflorescence ethyl acetate extract against RAW264.7 LC50 = 1 073.16μg/mL. Both Candida species and Mycobacterium species had their minimum inhibitory concentra-tions (MIC) tested using the minimum inhibitory concentration testing method. In the study Candida auris, Candida glabrata, Candida tropicalis and Candida parapsilosis were used to evaluate both the use of modified isoniazid derivatives as repurposing agents and the Cannabis indica/sativa extracts. The derivatives showed activity that could consider them probable treat-ment agents against candidiasis caused by non-Candida albicans Candida species. Compounds such as N’-(bis (2-hydroxyphenyl) methylene) isonicotinohydrazide · (salicylic acid) (IBS 008), N’-(propan-2-ylidene) isonicotinohydrazide (IBS 013) and N’- (propan-2-ylidene) ison-icotinohydrazide · (salicylic acid) (IBS 014), all showed activity against Candida auris at an MIC = 31.25μg/mL. Cannabis indica/sativa vegetative leaves of ultra-pure water extract also showed noteworthy results against Candida auris with an MIC = 62.5μg/mL. These results were confirmed by the performance of a flow cytometry assay where the parent-gated cells showed an apoptotic rate ≥80% for both Cannabis extracts and modified isoniazid derivatives. The Cannabis indica/sativa extracts were evaluated against the Mycobacterium species, the vegetative leaves of ethyl acetate extracts displayed the most noteworthy activity against both Mycobacterium smegmatis and Mycobacterium aurum with MIC = 31.25μg/mL. A phytochemical evaluation was also performed using the two dimensional gas chromatog-raphy – time of flight – mass spectrometry (GC x GC –TOF-MS). This analysis was able to highlight important compounds found in all six extracts, these compounds also have been found by previous studies to have medicinal properties such as anti-inflammatory, antibiotic and an-algesic activity. Some of the compounds analysed include commonly known Cannabis com-pounds such as Δ9-tetrahydrocannabinol, TMS derivative detected at 19:47,6min and canna-bi-chromene which was found in both leaves and inflorescence n-hexane extracts and were detected at the periods of 18:08,6min and 18:52,6min. Other compounds include Oxabenzo (f) cyclobut (cd) inden- 8-ol-1a-à,2,3,3a,8b-à,8c-à- hexahydro- 1,1,3a-trimethyl -6-pentyl- found at 18:35,7min and 4,8,12,16- Tetramethylheptadecan -4-olide detected at 18:39,8min. This study highlighted the importance of medicinal plants used in traditional practices with both vegetative leaves and mature inflorescence of Cannabis as sources of phytochemicals that aid in combating microbial growth. Furthermore, a unique discovery of repurposing isoniazid derivatives showed the potential inhibitory activity against Candida species and an enhanced activity against Mycobacterial species.