Experimental Methodology for Antimicrobial Screening of Medicinal Plants: A Critical Review of Extraction, Phytochemical Characterisation and Susceptibility Testing Approaches
Kadambini Das
Department of Botany, Babasaheb Bhimrao Ambedkar Bihar University, Muzaffarpur, Bihar, India.
S. Manjula
Department of Botany, Government First Grade College, Channapatna, Bangalore University, Bengaluru, Karnataka, India.
N. Anil Kumar
Department of Botany, SYTR Government Degree College, Madakasira, Affiliated to S. K. University, Ananthapuram, Andhra Pradesh, India.
Bhagwati Prashad Sharma
Department of Botany, Sidharth Government College, Nadaun, Himachal Pradesh, India.
Debangshu Agrahari
Jeevak Ayurved Medical College & Hospital Research Center, Chandauli, Uttar Pradesh, India.
Saraswati Majhi
Department of Life Sciences, Rama Devi Women's University, Bhubaneswar, Odisha, India and P.G. Department of Botany, Shailabala Women's Autonomous College, Cuttack Affiliated to Rama Devi Women's University, Bhubaneswar, Odisha, India.
Sweta Mishra *
Biodiversity and Conservation Lab., Ambika Prasad Research Foundation, Odisha, India.
Sanjeet Kumar
Biodiversity and Conservation Lab., Ambika Prasad Research Foundation, Odisha, India.
Manvi Malwal
Department of Botany, Post Graduate Government College for Girl63s 11 Chandigarh, India.
*Author to whom correspondence should be addressed.
Abstract
The rise of antimicrobial resistance has renewed scientific interest in medicinal plants as a source of novel antimicrobial leads, yet the methodological rigour applied to their laboratory evaluation remains highly variable across the published literature. This review critically examines the experimental workflow used in antimicrobial screening of medicinal plants, from plant selection and extraction through to phytochemical characterisation, in vitro susceptibility testing, anti-virulence and synergy assessment, mechanistic elucidation and safety evaluation. Conventional extraction techniques such as maceration, Soxhlet extraction and hydrodistillation are compared against modern approaches, including ultrasound-assisted extraction and green solvent systems based on natural deep eutectic solvents, with attention to yield, selectivity and preservation of thermolabile constituents. Qualitative phytochemical assays and chromatographic identification methods are discussed alongside their limitations for confirming bioactivity. The review evaluates the principal bacterial susceptibility testing formats, including disc diffusion, broth microdilution, agar dilution and bioautography, before turning to the distinct methodological demands of antifungal and antibiofilm testing, where reference standards developed by different standard-setting bodies do not always agree. Approaches for quantifying synergy between plant extracts and antibiotics, inhibition of bacterial quorum sensing, mechanistic studies of antimicrobial action, and cytotoxicity screening are considered in turn, illustrated with case studies drawn from several plant families. Recurring weaknesses identified across the literature include inconsistent reporting of extract concentrations, poor standardisation of inoculum size and growth media, limited use of appropriate positive and negative controls, and insufficient attention to reproducibility and selectivity. Recommendations are offered for methodological harmonisation to strengthen the translational value of future research in this field.
Keywords: Medicinal plants, antimicrobial screening, extraction methods, minimum inhibitory concentration, phytochemical analysis, quorum sensing, antibiotic resistance