Background and Objectives: Marjoram (Origanum vulgare) is an important medicinal plant belonging to the Lamiaceae family, widely cultivated in Mediterranean regions and Iran. The aerial parts of this plant are rich in essential oils and valuable phenolic compounds and, due to the presence of thymol, it has been extensively used in traditional medicine. Efficient and selective extraction of volatile and non-volatile compounds from plant matrices is often associated with limitations such as thermal degradation of chemical constituents, prolonged extraction time, high consumption of organic solvents, and the risk of degradation of sensitive biomolecules. Therefore, the development of green and non-thermal extraction approaches aimed at enhancing extraction efficiency while preserving bioactivity is of great importance for the food and pharmaceutical industries. Cold plasma pretreatment has recently emerged as a promising technique for improving the extraction of bioactive compounds from medicinal plants, and several studies have reported its effectiveness. The present study was conducted to investigate the effect of different sample preparation conditions during the cold plasma pretreatment stage on the extraction of bioactive constituents from the aerial parts of marjoram. Cold plasma, as a novel technology, has demonstrated a significant potential to enhance the extraction process through the generation of reactive oxygen and nitrogen species and the induction of microstructural modifications in the cell wall and cellular matrix, leading to increased tissue permeability and facilitating solvent penetration and the release of secondary plant metabolites.
Methodology: This study was conducted in a completely randomized design with four treatments and three replications. The treatments included dried powdered samples without solvent, dried unpowdered samples without solvent, powdered samples with solvent that were subjected to plasma pretreatment before extraction, and a control sample without plasma pretreatment. In this research, a dielectric barrier discharge (DBD) cold plasma system manufactured in Iran (Kavoshyaran Company, Enhanced Tech-18A model) was used. After treatment application, extraction was carried out using 70% methanol at a ratio of 5:1 (solvent volume to sample weight) by the maceration method. The antioxidant activity of the samples was evaluated using the free radical scavenging assay based on DPPH. Total phenolic content was determined using the Folin–Ciocalteu reagent, and the results were expressed as milligrams of gallic acid equivalent per gram of dry sample weight. The amounts of flavones, flavonols, and total flavonoids were determined by the colorimetric method using aluminum chloride reagent, with quercetin used as the standard. Total tannin content was determined using the colorimetric method based on vanillin–hydrochloric acid reagent, and the results were reported as milligrams of catechin equivalent per gram of dry sample weight. Total carbohydrate content was measured by the colorimetric method using anthrone reagent, with pure glucose as the standard. The data obtained from the measurement of different traits were analyzed using JMP software, and mean comparisons were performed using Tukey’s test.
Results: The results indicated that sample preparation conditions had a significant effect on most phytochemical attributes. The powdered samples treated with solvent exhibited the highest antioxidant activity (79.21%) and the greatest contents of flavones and flavonols (0.81 mg quercetin equivalents g⁻¹ dry weight), total phenolics (93.36 mg gallic acid equivalents g⁻¹ dry weight), and total flavonoids (10.37 mg quercetin equivalents g⁻¹ dry weight). In contrast, the lowest values for most compounds were observed in powdered samples without solvent. The addition of solvent increased antioxidant activity by 34.94% and total phenolic content by 26.37%. The highest tannin content (6.80 mg catechin equivalents g⁻¹ dry weight) was recorded in powdered samples without solvent, whereas the lowest value (3.41 mg catechin equivalents g⁻¹ dry weight) was observed in non-powdered samples without solvent, with no significant difference compared to the control and powdered samples with solvent. Unlike other measured traits, powdering the samples without solvent led to a 99.41% increase in tannin content. These findings suggest that powdering the samples in the presence of solvent during cold plasma pretreatment enhances the release and extraction of most secondary metabolites, while tannins respond differently.
Conclusion: The findings of this study demonstrated that cold plasma pretreatment combined with powdered sample preparation in the presence of solvent is the most effective approach for extracting secondary metabolites from marjoram extracts, including phenolic compounds, flavonoids, tannins, and total carbohydrates. This strategy can be considered a green and efficient method for producing plant extracts with high bioactivity. However, further optimization of plasma and extraction parameters, as well as evaluation of extract quality and stability, is required to facilitate industrial-scale applications. Overall, the results highlight that appropriate selection of sample preparation conditions in plasma-based processes plays a crucial role in determining the composition and quality of marjoram extracts.