Enrichment of novel antimicrobial compound from stereospermum fimbriatum using supercritical carbon dioxide extraction

Abstract

Stereospermum fimbriatum or “Chicha” (S. fimbriatum) is an undiscovered medicinal plant that is traditionally used for itchy skin, earache, stomachache and postpartum treatment. The present study was designed to evaluate antimicrobial properties of flowers, leaves, stem bark and twig of S. fimbriatum against eleven skin-associated pathogens as well as to isolate the bioactive compound from the active extract of S. fimbriatum and investigate its structure activity relationship by molecular docking. Supercritical carbon dioxide (sc-CO2) extraction was also carried out in order to obtain an enriched extract containing the isolated compound. The phytochemicals screening was done to determine the presence of alkaloids, terpenoids, flavonoids, steroids, saponins and tannins. Successive soxhlet extraction was conducted using n-hexane, dichloromethane (DCM) and methanol. Disc diffusion, minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) assays were done to examine the antimicrobial activity of the crude extracts. Bioassay-guided isolation of active compound was conducted by column and thin layer chromatographies. Molecular docking studies were done on five main targets for anti-MRSA activity, namely, penicillin binding proteins 2a, β-lactamase, DNA topoisomerase IV, dehydrosqualene synthase and sortase A. The binding sites and docking scores between a known inhibitor (positive controls) and an isolated compound were compared. Phytochemical screening revealed the presence of terpenoid, steroid, flavonoid, saponin and tannin in different parts of S. fimbriatum. The DCM extract of stem bark (DS) was found to be the most potent extract followed by n-hexane extract of the stem bark (NS) against Staphylococcus epidermidis, methicillin-resistant S. aureus (MRSA) and S. aureus with inhibition zones ranged from 13 to 16 mm. MIC values for NS and DS ranged between 400-800 µg/mL against S. epidermidis, MRSA and S. aureus. The bioassay-guided isolation of S. fimbriatum’s stem bark led to the discovery of a novel compound of Angucycline family, coded as C1 (11-hydroxy-8-methoxy-4,5-dimethyltetraphene-1,7,12(2H,4aH,12bH)-trione) with molecular formula of C21H18O5. MIC values of C1 were as low as 3.13 µg/mL to 6.25 µg/mL, against S. epidermidis, MRSA and S. aureus. Molecular docking of C1 indicated that it can be a good candidate for the development of a new anti-MRSA drug. The optimum condition for an enriched extract of sc-CO2 to have maximum recovery of C1 at targeted MIC value of 400 µg/mL was suggested to be operated at 40 °C and pressure at 30 Mpa, with addition of 6 % co-solvent (ethanol). Sc-CO2 extraction provided a low operational cost with less time and solvents consumption as compared to soxhlet extraction. Overall, the present study has accomplished to highlight the selectivity of sc-CO2 extraction for targeted bioactive compound as well as reveal the potential of S. fimbriatum as a candidate of antimicrobial agents for skin infections treatment, specifically, MRSA.