Origanum dictamnus L. is a medicinal plant known for its rich content in bioactive compounds. The plant cell wall consists of various structural polysaccharides such as cellulose, hemicellulose, and pectin, along with lignin, proteins, and bioactive compounds. These compounds are either trapped within the plant cell wall or free in the cytocol of the plant cell. Enzyme-assisted extraction (EAE) is a green technology that relies on the enzymes ability to selectively degrade the plant cell wall, thereby facilitating the release of bioactive compounds.

In the present study, EAE was applied to extract bioactive compounds from the leaves of Origanum dictamnus L. using the commercial enzyme preparation Cellic® CTec3 HS (Novozymes) and was compared to conventional extraction with ethanol–water mixtures of various concentrations. A Taguchi experimental design was applied to determine the optimal EAE conditions. The variables were enzyme loading (50, 100, and 200 U/mg), solid-to-liquid ratio (1, 4, and 7% w/v), and extraction time (1, 3, and 6 h). The responses were total phenolic content (TPC) and total flavonoid content (TFC). Conventional extraction was performed using ethanol–water mixtures of 0-100% v/v. TPC was determined using the Folin–Ciocalteu method and TFC with the aluminum chloride method. EAE achieved the highest TPC yield of 164.8 ± 5.2 mg GAE/g DW, at 1% w/v, 100 U/mg, and 6h. The maximum TFC yield reached 92.5 ± 5.7 mg CAE/g DW with 7% w/v, 100 U/mg, and 1h. In comparison, conventional extraction with 40% ethanol (v/v) gave the maximum TPC of 133.2 ± 0.5 mg GAE/g DW and TFC of 67.9 ± 0.9 mg CAE/g DW. These results support the potential of EAE as an efficient and sustainable method for the extraction of bioactive compounds from Origanum Dictamnus L.