Comparative Evaluation of Ethanol and Supercritical CO2 Extracts of Malaysian Stingless Bee Propolis for Oral Formulation: Phytochemical Profile, Antioxidative Property, and Gastrointestinal Digestive Enzyme Inhibition

Metabolic disorders such as type 2 diabetes mellitus and obesity are increasingly prevalent and pose major global health challenges. These conditions are characterized by dysregulated glucose and lipid metabolism, often accompanied by chronic inflammation and oxidative stress. One emerging therapeutic strategy involves the inhibition of key digestive enzymes, α-amylase, α-glucosidase, and lipase, in the gastrointestinal tract to reduce postprandial glucose and lipid absorption. Natural products offer a rich source of enzyme inhibitors and antioxidants, with propolis among the promising candidates due to its diverse phytochemical composition. Recent interest has focused on Malaysian stingless bee propolis for its potential use in oral formulations targeting metabolic dysfunction. However, the therapeutic efficacy of propolis is highly dependent on the extraction method used, which influences both chemical composition and bioactivity. Ethanol extraction is commonly used to obtain phenolic- and flavonoid-rich extracts, but it has drawbacks, including solvent residues and limited scalability. Supercritical carbon dioxide (SFE-CO2) extraction offers a greener, solvent-free alternative with tunable selectivity and improved preservation of heat- and light-sensitive compounds. This study aims to compare ethanol-extracted (EE-MP) and SFE-CO2–extracted (SFE-MP) Malaysian stingless bee propolis, evaluating their phytochemical profiles, antioxidant capacity, and in vitro inhibitory effects on key digestive enzymes.

The phytochemical profiles of EE-MP and SFE-MP were assessed by determining Total Phenolic Content (TPC), Total Flavonoid Content (TFC), and free radical scavenging activities via DPPH and ABTS assays. Inhibitory effects against α-amylase, α-glucosidase, and lipase were evaluated using standard in vitro enzyme assays.

Both extracts contained similar classes of bioactive compounds, including phenols, flavonoids, terpenoids, and glycosides. While EE-MP showed higher TPC and TFC, SFE-MP exhibited stronger DPPH radical scavenging activity (IC50 = 30.96 µg/mL vs. 35.64 µg/mL), with comparable ABTS results. Enzyme inhibition assays revealed that both extracts significantly inhibited digestive enzymes, with SFE-MP demonstrating greater α-amylase inhibition (61.50% vs. 56.06%) and superior lipase inhibition (91.61% vs. 88.62%) than that of EE-MP, surpassing even orlistat (72.11%).

The enhanced bioactivity observed in SFE-MP, despite its lower phenolic and flavonoid levels, suggests that SFE-CO2 extraction may selectively retain compounds with higher functional potency. Its stronger antioxidant performance and greater inhibition of digestive enzymes highlight the importance of extraction efficiency over total phytochemical yield. Moreover, the cleaner extraction profile of SFE supports its relevance for developing formulations that prioritize purity and stability. These findings indicate that extraction methodology plays a critical role in shaping the therapeutic potential of propolis.

This study demonstrates that both EE-MP and SFE-MP exhibitmeaningful antioxidant and enzyme-inhibitory activities relevant to the management of metabolic disease. While each extraction method offers distinct advantages, SFE-CO2 provides a viable path toward producing high-purity extracts suitable for oral applications. Overall, the results support Malaysian stingless bee propolis as a promising natural resource for future development of metabolic health interventions.