In vitro evaluation of antibacterial, antibiofilm, and embryotoxicity profiles of individual and combined Moringa oleifera and Hygrophila auriculata leaf extracts against Proteus mirabilis

The increasing resistance of pathogenic bacteria to antibiotics has driven interest in medicinal plants such as Moringa oleifera ( M. oleifera ) and Hygrophila auriculata ( H. auriculata ), which are inexpensive, traditionally accepted, and known for their antimicrobial, antioxidant, and nephroprotective properties. This study evaluates the antibacterial, antibiofilm, and cytotoxic potential of aqueous leaf extracts of M. oleifera and H. auriculata, individually and in combination against Proteus mirabilis . Leaf extracts were prepared using four different methods: boiling, steam extraction, ayurvedic decoction, and 50% ethanolic Soxhlet extraction. Antibacterial activity was assessed using agar well diffusion, Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) assays. Biofilm inhibition was quantified using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Embryotoxicity was assessed using zebrafish embryos toxicity assay. The chemical composition of the extracts was characterized using Fourier-transform infrared spectroscopy (FTIR) and gas chromatography-mass spectrometry (GC-MS). The combined treatment of H. auriculata (decoction) and M. oleifera (boiled) exhibited enhanced antibacterial and antibiofilm efficacy, with reduced MIC, MBC, and MBEC₅₀ values (0.1220- 0.4883 mg/mL; 0.2441-0.9766 mg/mL; 0.6098 - 2.161 mg/mL) compared to individual treatments. When applied separately, leaf extracts of M. oleifera showed MIC, MBC, and MBEC₅₀ ranges of 0.2441 - 0.9766 mg/mL, 0.4883-1.9531 mg/mL, and 1.229-4.227 mg/mL, respectively, while H. auriculata exhibited ranges of 0.4883-0.9766 mg/mL, 0.9766-1.9531 mg/mL, and 1.029-3.322 mg/mL, respectively. Zebrafish embryo toxicity assays revealed a dose-dependent response; however, the combined treatment exhibited reduced embryotoxicity, with the highest LC₅₀ value (1812.74 µg/mL) compared to M. oleifera (1527.5 µg/mL) and H. auriculata (1274.28 µg/mL) leaf extracts. FTIR analysis of the selected aqueous extracts and GC-MS analysis of the corresponding dried leaf materials provided preliminary information on their chemical characteristics and volatile/semi-volatile constituents, respectively. The study outlines promising antimicrobial and antibiofilm potential of leaf extracts of M. oleifera and H. auriculata , particularly when used in combination. Their enhanced antimicrobial activity and potential combined interaction, together with low embryotoxicity levels, suggest that these leaf extracts warrant further investigation as alternative therapeutic candidates for managing infections caused by P. mirabilis.