Extraction of Bioactive Compounds from Microalgal Feedstock and Its Derivatives: A Comparative and Integrative Approach
Abstract
Spirulina platensis, known for its wealth of proteins, pigments, and bioactive compounds, plays a crucial role in nutritional and therapeutic applications. Its bioactive components, particularly chlorophylls and pheophytins, are valued in nutraceutical, pharmaceutical, and agricultural sectors for their photosensitizing properties that can induce oxidative damage in pests by generating reactive oxygen species. However, there is a need to refine extraction methods to enhance the yield and selectivity of these compounds. This thesis provides a comprehensive comparison of traditional and modern extraction methods, moreover an advanced approach using an integrated alternative solvent and supercritical carbon dioxide (SCCO₂) sequential extraction was developed in this research. Additionally, the study explores the purification of semi-synthesized chlorophyllin, which typically contains 15-25% chlorin e6 and significant inactive components, underscoring the economic importance of developing cost-effective enrichment methods for these compounds. So, an additional objective of this thesis aims to optimize extraction processes and improve the purity and yield of chlorin molecules, contributing significantly to the field. An HPLC-MS method was established to accurately profile bioactive compounds, specifically targeting chlorophylls and their derivatives across various Spirulina extracts and purified chlorophyllin samples, this analytical technique was crucial for assessing the composition of these substances. Moreover, UV-spectrophotometry and thin layer chromatography were employed for compositional analysis. Additionally, all samples underwent biological activity evaluations including antioxidant activity and GST enzyme activity to ensure their efficacy for potential applications, while also providing a further understanding of their properties. For Spirulina platensis, the comparative analysis of extraction techniques showed that conventional solvent extraction assisted by ultrasonication using methanol and ethanol provided enhanced efficacy in extracting a wide range of polar and non-polar compounds. Conversely, non-polar solvents such as n-hexane were highly selective for hydrophobic compounds, notably beta carotene. Additionally, supercritical carbon dioxide (SCCO₂) extraction with modifiers including n-hexane and ethanol achieved an 18.7% increase in chlorophyll and an 88.2% increase in carotenoid yields compared to conventional ethanol extraction. Furthermore, utilizing the cascade process reduced organic solvent consumption by a factor of 40, underscoring its sustainability and efficiency. Biological activity assays showed that cascade SCCO₂ extraction, particularly when combined with n-hexane in the initial step, exhibited a high antioxidant activity of 118.5 mg/g ascorbic acid equivalent, reflecting superior antioxidant compound retention. In the Glutathione S-Transferase (GST) enzyme assay, the SCCO₂-CPME combination achieved the highest GST activity (0.03 µmol/mL/min), outperforming SCCO₂ alone (0.02 µmol/mL/min). On the other hand, the implementation of a binary co-solvent system comprising 20% ethanol and 80% aqueous TTAB (tetradecyltrimethylammonium bromide) in the developed integrated sequential extraction method yielded enhanced efficiency. This approach resulted in a 1.8-fold and 8.8-fold increase in total chlorophyll yield compared to pure TTAB and pure ethanol when used as co-solvents, respectively. Additionally, this system outperformed ultrasonication-assisted solvent extraction with TTAB and cascade SCCO₂ extraction with ethanol, delivering a 3.5-fold and 1.5-fold higher chlorophyll yield, while reducing ethanol consumption by 96% and extraction time by 47% compared to SCCO₂/ethanol. The synergistic action of ethanol and TTAB resulted in extraction with high antioxidant activity (9.9 mg/g ascorbic acid equivalent) and GST activity (0.2 μmol/mL/min), representing a 33.1% improvement over aqueous TTAB extraction, showing its suitability for bio pesticidal applications. For commercial chlorophyllin purification, the results indicated that conventional solvent extraction with methanol and ethanol achieved the highest chlorin e6 concentrations (0.13 mg/mL and 0.08 mg/mL, respectively), with Soxhlet extraction using methanol outperforming n-hexane by 11-fold. Additionally, SCCO₂ extraction employing a binary system of ethanol and SCCO₂ enhanced chlorin e6 yields compared to SCCO₂ alone, attributed to the polar nature of chlorin molecules. Biological activity assessments revealed superior antioxidant activity (96.2 mg ascorbic acid equivalent/g purified material) and GST activity (0.02 µmol/mL/min) for samples purified via methanol extraction. Overall, this research emphasizes environmental sustainability through reduced solvent use and shorter extraction times, offering a scalable and eco-friendly framework for processing Spirulina platensis. It also offers valuable insights into the purification of semi-synthesized chlorophyllin structures using various extraction techniques and underscores the importance of robust analytical methods for comprehensive chlorophyllin characterization and evaluation of its biological activities.