Integration of HPTLC with ATR-FTIR, HRMS and bioquantification for evaluation of antibacterial and antioxidant compounds in marine sponges

Marine sponges are an abundant source of unique bioactive compounds, offering immense potential for the development of innovative medicines. This study presents an analytical approach for the identification of bioactive compounds in marine sponge extracts. The approach capitalizes high-performance thin-layer chromatography (HPTLC) - Effect-Directed Analysis (EDA) with high-resolution mass spectrometry (MS) and Fourier-transform infrared (FTIR) spectroscopy, enabling identification of bioactive compounds. HPTLC-EDA was done to evaluate radical scavenging and antimicrobial activities against five pathogenic bacterial strains: Escherichia coli, Klebsiella pneumoniae, Bacillus spizizenii, Staphylococcus aureus, and methicillin-resistant Staphylococcus aureus (MRSA). The bioquantification of streptomycin and gallic acid was conducted using image analysis of HPTLC chromatograms, with subsequent analysis via antimicrobial assays against the aforementioned bacterial strains and DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging assays. The developed HPTLC chromatograms, utilizing toluene:ethyl acetate:acetic acid (60:37.5:2.5, V/V/V) as a mobile phase for the first time, enabled the optimal separation of bioactive compounds from complex marine sponge extracts. Avarol from Dysidea avara was identified as the compound with the highest potential using FTIR and HRMS techniques. The study highlights a high-throughput, cost-effective approach for screening bioactive metabolites from marine sponges using HPTLC-bioassay. This approach accelerates therapeutic discovery and offers a sustainable, eco-friendly alternative to traditional methods.