Comparative Analytical Study of Propolis Extracts Using Aqueous, Aqueous–Organic Vinegar, and Ethanolic Methods: Development of a Green Ultrasound-Assisted Extraction (AOVPE). [Patent No. 1969]

Background Propolis, a bioactive resin produced by Apis mellifera, is widely recognized for its antimicrobial, antioxidant, and anti-inflammatory properties. Conventional extraction methods rely heavily on ethanol, which may limit its applicability in food-grade and medical formulations. Recently, green extraction approaches have gained attention, particularly those utilizing food-safe solvents such as organic acids. The incorporation of organic vinegar (acetic acid-based solvent) into aqueous systems represents a promising alternative to enhance extraction efficiency and broaden the spectrum of bioactive compounds. Objective This study aims to develop and evaluate an innovative aqueous–organic vinegar extraction method (AOVPE) assisted by ultrasound, and to compare its chemical composition with conventional aqueous (APE) and ethanolic (EPE) propolis extracts. Methods Raw propolis was extracted using three different solvent systems: APE: distilled water AOVPE: mixture of apple cider vinegar and water (10:1, v/v) EPE: ethanol-based extraction AOVPE Extraction Protocol (Innovation Core) Crushed raw propolis was macerated in a water–vinegar mixture under dark, warm conditions Ultrasound-assisted extraction at 85°C for 6 hours Sequential filtration and cooling (<4°C) to remove wax impurities Re-filtration to obtain a clear, particle-free extract Chemical Analysis GC-MS for identification of volatile and semi-volatile compounds Quantitative comparison of 8 major compounds across extracts Comparative profiling of fatty acids, terpenes, and phenolic derivatives Results Comparative Composition Analysis The AOVPE method (water + vinegar) demonstrated superior extraction efficiency for most compounds: Highest levels observed in AOVPE for: Isopimaric acid (26.88%) Androstan derivatives (12.71%) Docosa-diyn-diol (12.05%) Thunbergol (7.82%) Farnesol (1.57%) Compounds absent in ethanol extract (EPE) but present in AOVPE: Terpenoids Steroidal compounds Complex alcohol derivatives APE showed moderate extraction efficiency EPE was selective but limited in compound diversity Discussion The results confirm that the addition of organic vinegar (acetic acid) significantly enhances extraction efficiency compared to water alone. The acidic environment: Improves solubility of both polar and semi-polar compounds Facilitates the release of resin-bound bioactives Enhances stability of certain phenolic and terpenoid compounds The synergy between water (polar solvent) and vinegar (weak organic acid) creates a dual-action extraction system capable of dissolving a broader spectrum of compounds than ethanol alone. Unlike ethanolic extraction, AOVPE: Avoids toxic or flammable solvents Produces a food-grade, biocompatible extract Preserves compounds that may degrade or remain insoluble in ethanol This positions AOVPE as a green, sustainable, and scalable extraction technology suitable for pharmaceutical, nutraceutical, and functional food applications. Conclusion The aqueous–organic vinegar extraction method (AOVPE) represents a significant advancement in propolis processing. By explicitly incorporating apple cider vinegar as a functional extraction solvent, this method achieves: Enhanced extraction efficiency Higher chemical diversity Improved biological safety It outperforms both aqueous and ethanolic methods, making it a strong candidate for industrial and medical applications.