Document Type : Research Paper
Authors
1 M.Sc. student, Horticulture Department, Agriculture Faculty, Urmia University, Urmia, Iran
2 Horticulture Department, Agriculture Faculty, Urmia University, Urmia, Iran
3 Soil and Water Research Department, West Azerbaijan Agricultural and Natural Resources Research and Education Center, Agricultural Research, Education and Extension Organization (AREEO), Urmia, Iran
Abstract
Background and Objective: Glycyrrhiza glabra L. (licorice) is a widely recognized medicinal plant known for its diverse biological properties and rich content of bioactive compounds, including glycyrrhizic acid, phenolics, and flavonoids, which contribute significantly to its antioxidant activity. Efficient extraction of these compounds is essential to fully utilize the plant’s potential. This study aimed to optimize the extraction conditions for these bioactive constituents from licorice leaves using ultrasound-assisted extraction (UAE). The primary objective was to determine the optimal conditions for maximizing the yield of bioactive compounds.
Methodology: Licorice leaves were collected, dried, and ground into a fine powder. Extraction was carried out using ultrasound-assisted extraction (UAE) based on a central composite design (CCD) and response surface methodology (RSM). The effects of three independent variables, temperature (X₁: 22.77–75.22 °C), extraction time (X₂: 1.59–18.40 min), and solvent concentration (X₃: 22.77–75.22%), were evaluated at five levels within predefined ranges to determine their impact on the extraction efficiency of selected phytochemicals. The extraction experiments were performed in 20 runs using an ultrasonic device operating at 120 Hz. The resulting extracts were analyzed for glycyrrhizic acid (GA) content using vanillin reagent with absorbance measured at 535 nm, total phenolic content (TPC) using Folin–Ciocalteu reagent at 760 nm, total flavonoid content (TFC) using aluminum chloride solution at 415 nm, and antioxidant activity via the DPPH radical scavenging assay at 517 nm. All spectrophotometric analyses were performed using a UV-2100 PC spectrophotometer. Statistical analysis, model fitting, and optimization of the extraction parameters were conducted using Design Expert version 10 software.
Results: Statistical analysis showed that a second-order regression model provided the best fit for glycyrrhizic acid (GA), total phenolic content (TPC), and DPPH radical scavenging activity, while an interaction model best described total flavonoid content (TFC). All models were statistically significant (P < 0.05), and non-significant lack-of-fit tests indicated good model adequacy. The R² values ranged from 0.745 to 0.798, demonstrating strong explanatory power. Among the factors evaluated, extraction time (B) had the most significant effect on GA (P < 0.05). For TPC, the temperature–time interaction (AB) showed the greatest positive effect (coefficient: 28.41), while solvent concentration (C²) had a highly significant negative effect (coefficient: –25.77). In TFC, the temperature–time interaction (AB) exerted the strongest positive influence (coefficient: 18.13), whereas extraction time (B) had a negative effect. For DPPH radical scavenging activity, solvent concentration (C²) was the most influential variable (coefficient: –16.51), with the temperature–time interaction (AB) also highly significant (coefficient: 17.15). Three-dimensional response surface plots confirmed the complex interactions among variables and highlighted optimal extraction points for each response. Overall, higher temperatures and longer extraction times enhanced TPC and flavonoid yields, although excessive conditions could cause degradation. Solvent concentration critically affected extraction efficiency, with moderate concentrations generally outperforming both very low and very high levels. Based on these results, the optimal extraction conditions were estimated to be a temperature of 57–70 °C, an extraction time of 35–45 minutes, and a solvent concentration of 50–60%.
Conclusion: This study demonstrated that the simultaneous optimization of key extraction variables, including solvent concentration, extraction time, and temperature, using response surface methodology (RSM) significantly improves the efficiency of extracting glycyrrhizic acid, phenolic compounds, flavonoids, and antioxidant activity from licorice leaves. The results emphasized the critical roles of temperature and extraction time in determining yield. The use of RSM, as a robust statistical tool, allowed the identification of optimal conditions with a minimal number of experimental runs. These findings can be applied to optimize industrial extraction processes for licorice secondary metabolites.
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