Biochemical Characterization and In Vitro Testing of Extracts

This module explores the journey from plant material to the evaluation of bioactive compounds, integrating techniques in natural product extraction, biochemical analysis, cell culture, and enzymatic pathway inhibition.

First, students will be introduced to plant extracts-concentrated substances derived from various plant parts, such as leaves, stems, roots, flowers, and seeds. Multiple extraction techniques will be presented, each with its own advantages and limitations. The selection of an appropriate extraction method will be discussed in relation to the plant material used, the target compounds, the desired level of purity, and the intended application of the final extract.

Next, the biochemical characterisation of the extracts will be demonstrated using three analytical assays: the Folin–Ciocalteu method for total phenolic content, the aluminium chloride assay for flavonoid content, and the FRAP assay for the evaluation of antioxidant activity. Data obtained from these assays will be subjected to thorough analysis and interpretation.

The third section will focus on basic in vitro cell culture techniques using human cell lines to investigate the cytocompatibility of the extracts. Students will perform the MTT assay, a well-established method for assessing cytotoxicity, using a range of extract concentrations and incubation periods. Subsequent data analysis will be conducted to determine a safe concentration range for further application.

Finally, the Renin-Angiotensin System (RAS) will be examined as a key regulator of blood pressure through an enzymatic cascade. Emphasis will be placed on the inhibition of angiotensin-converting enzyme (ACE) as a therapeutic target. Students will explore the physiological role of the RAS and screen plant extracts for potential ACE inhibitors. A SWOT analysis of existing screening methods will guide the development of informed recommendations.

By integrating natural product research with biomedical applications, this module offers a comprehensive and interdisciplinary learning experience.

General learning outcomes

• Formulate and apply appropriate methodological approaches for the extraction and screening of plant-derived compounds using biochemical and cellular assays.
• Select suitable in vitro bioassays (e.g. MTT, antioxidant assays, and enzymatic inhibition tests) for the identification and evaluation of bioactive fractions.
• Perform and interpret biochemical assays, including the Folin–Ciocalteu method, the aluminium chloride method, and the FRAP assay, for the characterisation of plant extracts.
• Apply basic cell culture techniques to assess the cytocompatibility of plant extracts using the MTT assay, and analyse data to determine safe concentration ranges.
• Explain the function of the Renin–Angiotensin System (RAS) and the role of angiotensin-converting enzyme (ACE) in blood pressure regulation.
• Design and implement screening strategies to identify potential ACE-inhibitory compounds in plant extracts.
• Critically evaluate and compare screening methodologies using SWOT analysis and formulate recommendations for methodological optimisation.

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