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 Researcher (Ph.D.), 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 play a significant role in its antioxidant activity. Efficient extraction of these compounds is essential to fully harness the plant's potential. This study aimed to optimize the extraction conditions of 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 powder. Extraction was performed 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 minutes), and solvent concentration (X₃: 22.77–75.22%)—were investigated at five levels within predefined ranges to assess their impact on the extraction efficiency of selected phytochemicals. The extraction process was conducted in 20 experimental runs using an ultrasonic device operating at 120 Hz. The resulting extracts were analyzed for glycyrrhizic acid (GA) content using a vanillin reagent and absorbance measurement at 535 nm, total phenolic content (TPC) using the Folin–Ciocalteu reagent and absorbance at 760 nm, total flavonoid content (TFC) using aluminum chloride solution and absorbance at 415 nm, and antioxidant activity via the DPPH radical scavenging assay with absorbance measured 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 carried out using Design Expert version 10 software.
Results: Statistical analysis revealed that second-order regression model provided the best fit for GA, TPC, and DPPH, while an interaction model best described TFC. All models were statistically significant (P < 0.05), and the lack-of-fit tests were non-significant, indicating good model adequacy. The R² values ranged from 0.745 to 0.798, suggesting a strong capacity of the models to explain the variability in the data. Among the evaluated factors, extraction time (B) had the most significant influence on GA (P < 0.05). For TPC, the interaction between temperature-time (AB) showed the greatest positive effect, with a coefficient of 28.41, and the effect of solvent concentration (C2) was highly significant (coefficient: -25.77). In the case of TFC, temperature-time interaction (AB) had the strongest positive impact (coefficient: 18.13), while time (B) exhibited a negative effect. Regarding DPPH radical scavenging activity, solvent concentration (C2) was the most influential variable (coefficient: -16.51), and the interaction between temperature and time (AB) was again highly significant (coefficient: 17.15). Three-dimensional surface plots confirmed the complex interactions between variables and the presence of optimal extraction points for each response. Overall, higher temperatures and longer extraction times enhanced the yields of TPC and flavonoids; however, excessive levels could lead to degradation. Solvent concentration also played a critical role in the extraction efficiency, with moderate concentrations generally outperforming both very low and very high levels. Based on the results, the optimal extraction conditions were suggested to be the temperature of 57–70°C, 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 RSM significantly enhances the efficiency of extracting glycyrrhizic acid, phenolic compounds, and flavonoids, as well as the antioxidant activity from licorice leaves. The results highlighted the critical role of temperature and time, in the extraction process. The application of RSM, as a robust statistical tool, enabled the identification of optimal conditions with a minimal number of experimental runs. This study finding could be considered in optimizing industrial extraction processes of licorice secondary metabolites.
Keywords
Main Subjects
)