Introduction

Glycyrrhiza glabra L., known as Liquorice, is one of the therapeutic herbs. Liquorice belongs to the Fabaceae family and has been recognized since ancient times for its ethnopharmacological values. Liquorice root contains different phytocompounds, such as glycyrrhizin, phenolic compound, glabrin A and B, and flavonoid, that have demonstrated various pharmacological activities. Pharmacological experiments have demonstrated that different extracts and pure compounds from this species exhibit a broad range of biological properties, including antibacterial, antiviral, anti-inflammatory, antioxidant, and antidiabetic activities1,2,3 Besides its medical purposes, Liquorice is a naturally occurring sweetener and ingredient used in the food sector4,5. The first stage in separating bioactive components from natural plant sources is extraction. Researching bioactive components from therapeutic plants has historically and continues today and has presented difficulties. Various extraction techniques have been devised to separate intended medicinal bioactive components to maximize production and reduce the loss of efficacy. Standard extraction techniques aim to extract the bioactive components while removing inert elements. Thus, the quality of the final product depends much on the best way of extracting the components6,7. To achieve optimal extraction results, using a method that effectively breaks down cell walls is essential, as it allows the release of intracellular compounds and maximizes the yield of valuable products8. Large amounts of solvent, long extraction durations, low yields, and high temperatures provide difficulties for conventional solvent-based extraction techniques like those used to extract Glycyrrhizic acid from Liquorice roots9.

Green extraction methods emphasize the importance of sustainable practices aimed at lowering energy consumption, reducing waste, and optimizing the use of natural resources. These environmentally friendly techniques prioritize the use of safer solvents and ensure responsible handling of residues to safeguard both human health and the environment10,11. Many new technologies are being studied to find the best way to extract bioactive compounds in larger amounts and of higher quality. In particular, Ultrasonic extraction is more efficient than traditional techniques, using less energy, solvents, and time. It’s considered environmentally friendly because it reduces energy and solvent use, improves extraction quality and yield, and takes less time12,13,14,15. Cold plasma has become more intriguing as a non-thermal extraction technique because of its simplicity, low cost, and high efficiency16. cold plasma is a versatile technique used in the food industry. It involves ionizing gas to create a mixture of excited atoms, ions, molecules, and radicals. The specific reactive species produced depend on the gas used to generate the plasma. Common gases include air, argon, helium, nitrogen, and mixtures of these gases17,18,19. The cold plasma technique Cold plasma can change the physical structure of the plant material, creating things like cracks and dents. This makes it easier for the desired compounds to be released, which leads to a higher extraction yield and a better choice for extraction than traditional methods because it can preserve important plant parts without damaging the plant’s structure20.

Despite advancements in extraction technologies, the combined use of UAE and NTP for isolating bioactive compounds from plants remains unexplored. Integrating these two methods can synergistically enhance extraction by disrupting cell structures and improving surface properties, leading to higher yields. This approach aligns with green extraction principles by reducing energy consumption and solvent use, offering a sustainable alternative to traditional methods. UAE improves mass transfer, while NTP enhances cell permeability, making their combination an efficient solution for overcoming extraction limitations. This work aims to establish and optimize a sustainable, high-efficiency extraction approach by harnessing the synergistic effects of ultrasonic and cold plasma techniques to improve the yield and quality of bioactive compounds extracted from Liquorice extract.

Materials and methods

Chemicals and reagents

All reagents used were of analytical grade. Distilled water served as the solvent for all extractions. Key chemicals, including Folin-Ciocalteu reagent, Sodium carbonate, Aluminum chloride, Potassium acetate, Methanol, Gallic acid, and DPPH, were obtained from Sigma-Aldrich (Germany). Quercetin and Glycyrrhizic acid standards were sourced from Merck (Germany).

Preparation of samples

The liquorice roots were carefully washed to eliminate any impurities and air-dried at room temperature (25 °C) in a shaded area to preserve the bioactive compounds. The drying process continued until the moisture content of the roots was reduced to around 10–12%. After drying, the roots were cut into small pieces and ground into a fine powder using a laboratory mill. The resulting powder was then passed through an 80-mesh sieve to achieve a uniform particle size, ensuring its optimal suitability for extraction processes.

Design of the experiment

The experimental design employed Response Surface Methodology (RSM) utilizing a Box-Behnken design with three central repetitions. The experimental design and optimization were conducted via Design Expert software (version 11.1.1.0, Stat-Ease, Inc., Minneapolis, USA).

Methods of extraction

Maceration extraction

For maceration extraction, 10 g of powdered Liquorice root was combined with 100 mL of distilled water. The amalgamation was heated to 50 °C and agitated for 60 min. The resultant solution was filtered with Whatman No. 1 filter paper and subsequently concentrated under decreased pressure at 37 °C with a rotary evaporator (IKA RV 10, IKA-Werke GmbH & Co. KG, Staufen, Germany). The extract was desiccated in an incubator (Memmert, Schwabach, Germany) at 37 °C21.

Ultrasonic-assisted extraction

Ultrasonic extraction was performed via an ultrasonic homogenizer (UP400-A, Hielscher Ultrasonics GmbH, Teltow, Germany). 10 g of Liquorice root powder was combined with 100 mL of distilled water and exposed to ultrasonic at 50 °C, Frequency 42 ± 5% kHz. Based on the experimental design Table 1. The extract was filtered, concentrated, and prepared as described in maceration extraction22.

Table 1 The effect of independent variables of cold plasma -ultrasonic on the quality properties of liquorice extract.
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Cold plasma-assisted extraction

Cold plasma extraction was conducted via a dielectric barrier discharge (DBD) plasma setup. The plasma configuration had two coaxial electrodes: a center wire electrode and a ring-shaped electrode positioned at a constant distance apart. The dielectric material used was a slender tube with an external electrode affixed to its surface. The high-voltage AC power source linked to the electrodes facilitated adjustable voltage (0–20 kV) and frequency (6–20 kHz). Argon gas served as the input gas, with a flow rate of 3 standard liters per min (SLM). The separation between the sample and the plasma nozzle was maintained at 2 cm, with the temperature at 50 ± 5 °C. The ocean Optic spectrometer (HR2000 + CG, Ocean Insight, Inc., Orlando, Florida, USA) was used to identify the different species created by the plasma. The spectrometer measured light wavelengths between 200 and 1000 nanometers, and used a fiber optic cable to connect the plasma to a computer, allowing the light spectrum to be analyzed23. The schematic of cold plasma is shown in Fig. 1.

Fig. 1
figure 1

Schematic of cold plasma production device.

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For cold plasma-assisted extraction, After mixing the Liquorice root powder with distilled water at a ratio of 1:10, the mixture was subjected to cold plasma (Laboratory Reactor); The duration of plasma therapy ranged from 1 to 10 min. The extract was filtered, concentrated, and prepared as described in the maceration method.

Ultrasonic-cold plasma extraction integration

The combination extraction approach involved consecutive ultrasonic and cold plasma treatments in a specially built reactor (Fig. 2). The independent variable was time, measured from 1 to 9 min. The samples underwent combined extraction, and the extracts were processed according to the previously outlined protocols. Table 1 enumerates the independent factors and their impacts on extract quality.

Fig. 2
figure 2

Schematic of the combined ultrasonic-cold plasma method.

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Assessment of extract quality

Determining the extract content

The content of the extract was calculated by weighting the amount extracted from 100 g of dry Liquorice root powder (Eq. 1).

$$\:\text{E}\text{x}\text{t}\text{r}\text{a}\text{c}\text{t}\:\text{c}\text{o}\text{n}\text{t}\text{e}\text{n}\text{t}\:\:\left(\text{\%}\right)=\left(\frac{{W}_{1}}{{W}_{2}}\right)\times\:100$$
(1)

where W1: Weight of the recovered extract (g) and W2: Weight of the plant material (g).

Total phenol content (TPC)

The total phenol content was quantified via the Folin-Ciocalteu colorimetric method, employing gallic acid as the standard reference. 0.01 g of dried extract was solubilized in 10 mL of 60% methanol. Subsequently, 0.1 mL of the solution was combined with 0.5 mL of 10% Folin-Ciocalteu reagent and 0.4 mL of 7.5% sodium carbonate. The reaction was permitted to continue in darkness for 30 min, after which absorbance was assessed at 765 nm utilizing a UV-Vis spectrophotometer (Unic 2100, Instruments Co., Ltd., Shanghai, China). The results were presented as mg of gallic acid equivalents (GAE) per g of dry extract24.

Total flavonoid content (TFC)

The total flavonoid content was assessed using the aluminum chloride technique. 0.01 g of dried extract was solubilized in 10 mL of 60% methanol. 0.1 mL of this solution was combined with 0.5 mL of 2% aluminum chloride and 3 mL of 5% potassium acetate. Following a 40 min incubation in darkness, the absorbance was measured at 415 nm. A quercetin standard was utilized, and the results were reported as mg of quercetin equivalents (QE) per g of dried extract24.

Glycyrrhizic acid content

Glycyrrhizic acid content was quantified by a high-performance liquid chromatography system (UltiMate 3000, Thermo Scientific, Inc., Waltham, Massachusetts, USA) equipped with a Diode Array Detector with detection occurring at 254 nm. Separation was performed on a C18 column (4.6 × 300 mm, 10 μm). Throughout the separation, the flow rate was 1mL/min, and the mobile phase consisted of water and acetic acid at a ratio of 6:60:34. The content of Glycyrrhizic acid was determined using a calibration curve established with Glycyrrhizic acid standards25 (Fig. 3).

Fig. 3
figure 3

The chromatogram of Glycyrrhizic acid of sample.

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Antioxidant activity

The antioxidant efficacy of the extracts was assessed using the 2,2-diphenyl-1-picrylhydroazyl (DPPH) radical scavenging technique. Various quantities of the extracts were formulated and combined with 1 mL of 90 µM DPPH solution. The mixes were incubated in darkness for 30 min, and absorbance was assessed at 517 nm. The IC50 value was ascertained, indicating the quantity necessary to control 50% of DPPH radicals26. Antioxidant activity calculated using Eq. (2):

$$\:\text{I}\text{n}\text{h}\text{i}\text{b}\text{i}\text{t}\text{i}\text{o}\text{n}\:\left(\text{\%}\right)=\left(\frac{({\text{A}}_{\text{B}}-{\text{A}}_{\text{A}})}{{A}_{B}}\right)\times\:100$$
(2)

where, AA absorbance values of extracts and AB absorbance values of the control (DPPH and methanol as the control no extract).

Optimization and statistical analysis

Using Response Surface Method (One-Factor), and Design-Expert software (Design Expert v11.1.1.0, Stat-Ease, Inc., Minneapolis, USA), the ultrasonic, cold plasma, and combination methodologies optimized the independent variables of the extraction process. The independent variables comprised extraction time, ultrasonic power, and the argon-to-air ratio in the plasma system. The dependent variables included yield of extract, TPC, TFC, and antioxidant activity. In the optimization process using the response surface method, the primary goal was to minimize the extraction time while maximizing the concentration of compounds. Specifically, the yield, TPC, TFC, and antioxidant activity were treated as dependent variables to be maximized, ensuring that the extraction process was efficient in both quantity and quality of the bioactive compounds. The experimental data were examined utilizing a second-order polynomial equation, and the ideal circumstances were determined. The models were validated through supplementary experiments conducted under the anticipated ideal conditions. Statistical analysis was conducted utilizing SPSS 16.0 software (SPSS Inc., Chicago, USA), and the differences between the techniques were assessed using LSD tests at a 1% significant threshold.

Comparison of extraction techniques

The ideal extraction parameters for each method were evaluated. The methods were evaluated and graded utilizing the Technique for Order of Preference by Similarity to Ideal Solution (TOPSIS). In this study, Microsoft Office Excel software (Microsoft Excel 2016, Microsoft Corp., Redmond, USA) was used for the Multi-Criteria Decision-making, and the TOPSIS. First, a decision matrix was constructed using the measured values for yield of extract, phenolic content, flavonoid content, glycyrrhizic acid content, and antioxidant activity as evaluation criteria. The matrix was then normalized to remove the influence of different units across criteria. Next, we assigned weights to each criterion according to its importance in the overall evaluation process, with greater emphasis on yield and bioactive content. The weighted normalized matrix was used to determine the positive ideal solution and negative ideal solution. The Euclidean distance of each extraction method from both the positive ideal solution and negative ideal solution were calculated. Finally, the relative closeness coefficient for each method was computed, indicating how close each extraction technique was to the ideal solution. Higher relative closeness coefficients signified better performance27.

Results and discussions

Optical emission spectroscopy of cold plasma

Figure 4 illustrates the spectrum derived from the argon and air plasma. The spectra of argon and air plasma at a voltage of 10 kV, within the wavelength range of 200–1000 nm, are presented. The spectra of argon atoms are observable within the 650–900 nm wavelength range. When plasma was created in the atmosphere, it interacted with different parts of the air. This created NOx in the UV spectrum, N₂ (336.9 nm), N⁺ (357.6 nm), N₂⁺ (375.4 nm, 380.3 nm), and highly reactive OH (305–312 nm). The atomic spectra of air constituents are observable within the 200–1000 nm wavelength range. The principal peaks associated with active oxygen and nitrogen species are discernible in the light emission spectra. The data indicated that the device produced fewer than 10 ppm of ozone during plasma treatment27.

Fig. 4
figure 4

The spectrum produced by the plasma produced in the (a) Ar, (b) Air.

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Extraction using the combined ultrasonic-cold plasma method

RSM statistical analysis of the effect of combined ultrasonic-cold plasma method on extract performance

The Box-Behnken experimental design was applied to evaluate the effects of three independent variables: argon gas-to-air ratio (A), ultrasonic power (B), and extraction time (C). A total of 15 experiments with five replications at the central point were conducted, and ANOVA was performed to analyze the main and interaction effects. Due to the meaningfulness of the models and the non-significance of lack of fit, the models have sufficient accuracy.

Results indicated that the argon gas-to-air ratio (A) and extraction time (C) significantly affected the quality of the extract, while ultrasonic power (B) significantly influenced the total flavonoid content (TFC) and extract percentage. The quadratic effects of the variables (A2) were significant at the 1% probability level for total phenolic content (TPC) and extract percentage, and at 5% for antioxidant activity. The quadratic effects of ultrasonic power (B2) and extraction time (C2) were significant for TPC, TFC, antioxidant activity, and extract percentage.

Furthermore, significant interaction effects were observed between the argon gas-to-air ratio and ultrasonic power (AB) on extract percentage, TPC, flavonoid content, and antioxidant activity. The interaction between time and argon-to-air ratio (AC) was significant only for antioxidant activity, while the interaction between ultrasonic power and time (BC) significantly affected extract yield, flavonoid content, and antioxidant activity (Table 2).

Table 2 The results of the statistical analysis of the response level for the extract extracted by the combined ultrasonic-cold plasma method.
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According to the coefficients of the obtained equations (Table 3), extraction time had the most significant effect on the quality characteristics. The effect was positive for both total phenolic content (TPC) and total flavonoid content (TFC), meaning that the compound amounts increased with time. Regarding the IC50 value, which reflects antioxidant activity, although the coefficient is negative, the inverse relationship indicates that antioxidant activity increases as IC50 decreases.

Table 3 The coded equation for the qualitative characteristics of the extract extracted by the combined ultrasonic-cold plasma method.
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The argon gas-to-air ratio positively influenced TPC but negatively impacted TFC and antioxidant activity. Ultrasonic power negatively affected TPC, TFC, and antioxidant activity. The interaction effect of time with gas ratio and power positively impacted TPC, but negatively affected TFC and antioxidant activity.

Figure 5 demonstrates that the models reliably predict experimental conditions. The model’s predictions closely align with the observed data, confirming that it effectively captures key variables and trends. This indicates that the model is well-calibrated, providing accurate predictions under the tested conditions. The consistency between predicted and observed values further supports the model’s robustness and applicability across various experimental scenarios.

Fig. 5
figure 5

Actual data versus Predicted data in (a) Yield of extraction, (b) Total phenol content, (c) Total flavonoid content, and (d) IC50.

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Figure 6a presents the three-dimensional diagram showing the effects of the argon gas-to-air ratio, ultrasonic power, and extraction time on the quality characteristics of the extract using the combined ultrasonic-cold plasma method. As shown in Fig. 6a,b, the yield of extract (%) increases with extraction time up to approximately 5 min, after which it decreases. Additionally, the argon gas-to-air ratio positively impacts extract yield, while increasing ultrasonic power leads to only minimal changes in extract percentage. The interaction between ultrasonic power and argon-air ratio up to 200 W and 50% argon gas showed a synergistic effect, but further increases in these variables led to a decrease in extract yield. Figure 6c,d demonstrate that the TPC increases with extraction time to about 5 min before decreasing. The TPC changes slightly with increased argon gas, while ultrasonic power positively affects phenolic compounds up to 200 W, after which it causes a decrease. The interaction of ultrasonic power and argon-air ratio at 200 W and 50% argon gas shows a synergistic effect for around 5 min, after which the effect declines. Figure 6e,f show an increase in TFC with increasing extraction time, but only a small increase is observed with higher argon gas-to-air ratio. Ultrasonic power reduces the TFC. The interaction of ultrasonic power and argon gas-to-air ratio leads to a decrease in TFC.

Figure 6g,h illustrate that the IC50 value decreases with increased extraction time, reflecting an increase in antioxidant activity. The increase in argon gas raises the IC50 value, reducing antioxidant activity. Regarding ultrasonic power, the IC50 value decreases up to 200 W, after which it increases slightly. Antioxidant activity improves up to 200 W and then declines.

Fig. 6
figure 6

The mutual effect of independent variables on qualitative characteristics in the extraction method using an ultrasonic-cold plasma reactor, (a,b) yield of extract (%), (c,d) total phenol, (e,f) total flavonoid, (g,h) antioxidant activity.

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Ultrasonic waves generate cavitation bubbles that disrupt plant cell walls, facilitating the release of intracellular compounds, including antioxidants, polyphenols, and essential oils. UAE is considered a greener extraction method, as it enhances extraction yields while using less solvent and operating at lower temperatures. Research has shown that UAE is effective in extracting phytoconstituents from various plants, such as Withania somnifera and Spondias tuberosa12,13,14,28. UAE has been proven to significantly increase the yield of phenolic compounds and flavonoids, thereby enhancing antioxidant activity in medicinal plants. Key extraction parameters like time, temperature, and solvent type are critical in optimizing the yield of bioactive compounds. For example, UAE has notably improved TPC in citrus peel extraction, where increased ultrasonic time and temperature led to higher TPC and improved antioxidant activity29,30,31. Ultrasound treatment induces two main effects: mechanical energy from cavitation and fluid flow caused by acoustic cavitation. These physical mechanisms create, expand, and collapse gas bubbles in the liquid, leading to cell wall disruption and the release of their contents. As extraction time increases, cavitation intensity intensifies, improving the yield of bioactive compounds. However, prolonged exposure to ultrasound may cause structural damage to the compounds due to oxygen exposure, free radicals, and denaturation. Optimizing extraction time can mitigate these negative effects, ensuring maximum yield without compromising compound integrity32,33,34,35,36,37.

Cold plasma, a non-thermal technology, creates reactive species like oxygen and nitrogen radicals that interact with the plant matrix to produce structural changes at the cellular level. This disrupts the plant’s surface, particularly its outer layers, releasing bioactive substances. Researchers have found that cold plasma makes it easier to get important oils out of plants like Foeniculum vulgare and Mentha spicata38,39,40. Several theories have been suggested to account for the improved extraction of active compounds after plasma treatment. One hypothesis proposes that plasma facilitates the release of bioactive compounds by disrupting cell walls while reducing potential damage from chemical extraction agents. Another perspective focuses on the reduction of surface tension, often attributed to hydrophobic compounds on plant surfaces, which can enhance the penetration and extraction of hydrophilic components41,42,43. Additionally, it has been suggested that reactive oxygen species (ROS) and reactive nitrogen species (RNS) generated during plasma treatment possess sufficient energy to initiate chemical reactions. These reactions can break covalent bonds within the cell wall matrix, thereby improving the efficiency of compound release from plant tissues44,45,46.

Studies have found that treating plants with cold plasma enhances the quantity of antioxidants and essential oils they possess44. The study by Ahmadian et al. (2023) demonstrates that Cold Plasma pretreatment enhances extraction efficiency by making plant surfaces more hydrophilic. This modification allows for better interaction between plant tissues and solvents, resulting in improved extraction yields47. In another study, Elmizadeh et al. (2023) reported that Cold Plasma pretreatment significantly increased the levels of antioxidants and total phenolics in extracts compared to conventional methods48.

Cold plasma treatment has been shown to improve the antioxidant properties of plant extracts, enhancing DPPH and ABTS scavenging activities. The antioxidant capacity is often correlated with the TFC, and optimizing the Ar/air ratio can maximize these effects49. The exposure duration of cold plasma significantly influences phenolic, flavonoid, and antioxidant activity during the extraction of medicinal plants. Studies indicate that cold plasma treatment enhances the bioactivity and extraction efficiency of numerous bioactive compounds, particularly phenolics, which are key to the antioxidant properties of botanicals50. Cold plasma interacting with plant products could induce structural changes improving the solubility and phenolic extraction51. Studies suggest that optimal exposure intervals can enhance flavonoid extraction. The reactive species generated during plasma treatment, such as ROS and RNS, carry enough energy to induce chemical reactions that can break covalent bonds in the plant cell wall structure52,53. Tests showing enhanced DPPH radical scavenging capacity in treated extracts suggest that cold plasma treatment is associated with increased antioxidant activity. However, exposure time is crucial; prolonged exposure may degrade sensitive compounds, requiring a balance to be struck Although cold plasma has shown promise in improving the extraction of beneficial compounds, excessive treatment can lead to the degradation of sensitive phytochemicals. Therefore, further research is necessary to optimize processing parameters for specific plant materials to balance both yield and quality54.

Cold plasma treatment has emerged as a promising method for enhancing the extraction efficiency of bioactive compounds, especially those with therapeutic properties. However, excessive exposure can lead to the degradation of these compounds, making it crucial to optimize treatment conditions to avoid such effects. Studies have shown that the argon-to-air (Ar/air) ratio plays a vital role in determining the phenolic and flavonoid content, extraction yield, and antioxidant activity of medicinal plants. Proper optimization of this ratio leads to extraction yields that surpass those of conventional methods, highlighting cold plasma’s efficacy as an advanced extraction technique53. Cold plasma treatment has also been found to significantly enhance the release of phenolic compounds, which are essential for antioxidant activity. It notably increases the levels of flavan-3-ols and flavonols in plant extracts, further emphasizing the potential of cold plasma to optimize the extraction of bioactive compounds, particularly phenolics29,30,31.

Combining cold plasma and ultrasonic techniques makes the best use of both technologies. Cold plasma alters the plant matrix chemically to improve permeability, whereas ultrasonic physically breaks down the cell walls. Researchers have found that this combination significantly increases the yield of bioactive components, such as phenolics and essential oils, compared to conventional extraction techniques55. The combination of ultrasonic and cold plasma techniques has shown the potential to improve the extraction of bioactive components from medicinal plants. This approach can enhance extraction efficiency while helping to preserve the integrity of the compounds. The combination approach is particularly useful for maintaining thermolabile chemicals, such as antioxidants, which degrade easily under thermal extraction techniques. Medicinal plants like ginseng (Panax ginseng) and G. glabra have been used to show that this combination extraction method works, greatly increasing the amounts of antioxidants and secondary metabolites56.

Optimizing of ultrasonic-cold plasma technique

After determining the effect of the variables of ultrasonic power, time, and the ratio of argon gas to air on extract extraction, optimization was done, and the optimal values ​​of the variables were determined. This optimization aimed to obtain conditions of independent variables (extraction time, ultrasonic power, and ratio of argon gas to air) that, at the optimal point, the maximum amount of TPC compounds, TFC, and percentage of the extract and the lowest value of IC50 be achieved The boundary conditions determined in the optimization were such that the variable values ​​of the extraction time were in the shortest time. The variables of the ultrasonic power and the ratio of argon gas to air were within the test range. A weight of one was given to all independent and dependent variables due to their equal importance. The results of the proposed optimization of 114.24 W power software were 6.17 min, and the ratio of argon gas combination with air was 91.71%.

For validation, the proposed points were evaluated in the laboratory. For this purpose, the values of the independent variables were rounded to the nearest whole number to be practical. Based on this, the ultrasonic power, extraction time, and ratio of argon gas with air were obtained as 114 W, 6.17 min, and 92%, respectively. The small difference between the theoretical optimum value and the experimental value obtained in the laboratory shows the correctness of the optimization method (Table 4).

Table 4 The results of the theoretical optimal value and the experimental value for the qualitative characteristic of the extract extracted by cold plasma.
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Glycyrrhizic acid content

After determining the optimal points in each extraction method, the amount of Glycyrrhizic acid content was measured at the optimal points. Table 5 shows the analysis results of extracts extracted at optimal points using different methods. According to the LSD test, the comparison of Glycyrrhizic acid content obtained from different extraction methods at their optimal points reveals distinct differences between the methods. The highest amount of Glycyrrhizic acid (118.64 mg/g extract) was achieved using the combined ultrasonic-cold plasma method, followed by the cold plasma method, and the ultrasonic method. In similar studies, the UAE method has consistently demonstrated superior efficiency in extracting bioactive compounds such as glycyrrhizic acid and glabridin from G. glabra compared to traditional extraction methods like maceration UAE facilitates a more rapid and efficient release of these compounds due to the ultrasonic cavitation effect, which enhances solvent penetration and disrupts plant cell walls, leading to higher yields57,58,59 .

Table 5 The amount of glycyrrhizic acid composition according to the optimal points of different extraction methods.
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The combined ultrasonic-cold plasma method showed a significant increase in the extraction of Glycyrrhizic acid compared to the individual ultrasonic and cold plasma methods, confirming the synergistic effect of combining these modern extraction techniques. When combined, UAE and cold plasma create a synergistic effect, maximizing extraction efficiency and further enhancing the process beyond the capabilities of each method individually60,61.

Comparing different extraction techniques

The results of the analysis of variance (ANOVA) showed a significant difference (p < 0.01) among the qualitative characteristics of the optimized extract obtained by different extraction methods (Table 6). The mean comparison between the optimal points of different methods is shown in Figs. 7, 8, 9, 10 and 11. As shown in Fig. 7, there is a significant difference between all methods in terms of the yield of extract (%) at the 1% probability level according to the LSD test. The highest amount was obtained from the cold plasma method, and the lowest amount was obtained from the maceration method. The yield of extract (%) increased by 1.24, 7.18, and 5.53% in ultrasonic, cold plasma, and combined ultrasonic-cold plasma methods, respectively, compared to the maceration method.

Table 6 The results of the analysis of variance (ANOVA) results for the qualitative characteristics of the optimized extract obtained by different extraction methods.
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Fig. 7
figure 7

The mean comparison between the optimal points of different methods on yield of Extraction (according to the LSD test, the means with a common letter do not have a significant difference at the 1% probability level, (P < 0.01)).

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Fig. 8
figure 8

The mean comparison between the optimal points of different methods on total phenol content (according to the LSD test, the means with a common letter do not have a significant difference at the 1% probability level, (P < 0.01)).

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Fig. 9
figure 9

The mean comparison between the optimal points of different methods on total flavonoid content (according to the LSD test, the means with a common letter do not have a significant difference at the 1% probability level, (P < 0.01)).

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Fig. 10
figure 10

The mean comparison between the optimal points of different methods on IC50 (according to the LSD test, the means with a common letter do not have a significant difference at the 1% probability level, (P < 0.01)).

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Fig. 11
figure 11

The mean comparison between the optimal points of different methods on Glycyrrhizic acid (according to the LSD test, the means with a common letter do not have a significant difference at the 1% probability level (p < 0.01)).

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In Fig. 8, the amount of TPC between all methods showed a significant difference at the probability level of 1% according to the LSD test. The highest amount was obtained from the cold plasma method, and the lowest amount was obtained from the maceration method. The total amount of TPC content increased by 10.23, 15.96, and 13.29% in the ultrasonic, cold plasma, and combined ultrasonic-cold plasma methods, respectively, compared to the maceration method.

In Fig. 9, the amount of TFC between all methods showed a significant difference at the probability level of 1% according to the LSD test. The two cold plasma and ultrasonic methods did not show any significant difference. The highest amount was obtained from the extraction with the help of ultrasonic-cold plasma, and the lowest amount was obtained from the maceration method. The amount of TFC in ultrasonic, cold plasma, and combined ultrasonic-cold plasma methods increased by 21.47, 22.19 and 42.41%, respectively, compared to the maceration method.

In Fig. 10, there was no significant difference in the IC50 value between all ultrasonic, cold plasma, and combined ultrasonic-cold plasma methods, but these methods showed a significant difference with the maceration method at the 1% probability level. The highest value of IC50 was obtained using the maceration method, which indicates the minimum antioxidant activity. The lowest value of IC50 was obtained using the extraction method with the help of ultrasonic-cold plasma, which indicates the maximum antioxidant activity. The value of IC50 in ultrasonic, cold plasma, and combined ultrasonic-cold plasma methods has decreased by 16.09, 14.08, and 18.31%, respectively, compared to the maceration method.

In Fig. 11, the amount of Glycyrrhizic acid content between all methods showed a significant difference at the probability level of 1% according to the LSD test. The highest amount was obtained from the ultrasonic-cold plasma method, and the lowest amount was obtained from the maceration method. The amount of Glycyrrhizic acid increased by 10.84%, 12.38%, and 15.89%, respectively, in ultrasonic, cold plasma, and combined ultrasonic-cold plasma methods compared to the maceration method. Modern extraction methods, such as UAE and cold plasma treatment, offer distinct advantages over traditional techniques like maceration. Unlike maceration, which is slow and often leads to low efficiency and compound degradation, UAE uses cavitation to rapidly break down cell walls, improving extraction efficiency. Cold plasma further enhances this by generating reactive species that modify cell walls, promoting the release of bioactive compounds. Together, these technologies provide faster, more efficient, and sustainable extractions with higher yields and better preservation of compound integrity60. The use of cold plasma as a non-conventional extraction technique is relatively new, and further studies are needed to better understand the optimal process conditions to be adopted. More in-depth research is also required to explore the mechanisms of plasma–plant matrix interactions, particularly to assess the possibility of side reactions that may occur in a highly oxidative environment, potentially generating hazardous substances. This understanding is crucial to ensure the feasibility of this technique at an industrial scale53.

Ranking of different extraction techniques by TOPSIS method

The maceration extraction method and optimal point of ultrasonic, cold plasma, and ultrasonic-cold plasma methods were compared and ranked using the multi-criteria decision-making and TOPSIS methods. According to Fig. 12, the ultrasonic-cold, cold, and ultrasonic methods have more efficiently extracted a better quality extract from Liquorice root than the maceration method.

Fig. 12
figure 12

Ranking of different extraction methods.

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The integration of UAE and cold plasma treatment offers a synergistic mechanism to enhance the extraction process by combining complementary physical and chemical actions that improve the release of bioactive compounds from plant tissues. The strengthening effect arises from the combined actions of these technologies: ultrasound mechanically disrupts cell walls through cavitation, while cold plasma chemically modifies them by generating reactive species. This dual mechanism enhances solvent penetration, mass transfer, and bioactive compound release, thereby increasing extraction efficiency and reducing processing time62,63,64.

Conclusion

This research investigated the combined ultrasonic-cold plasma extraction method for extracting bioactive compounds from Liquorice root. The hybrid approach significantly enhanced the extraction efficiency compared to traditional maceration and single-method techniques, resulting in increased antioxidant activity and higher concentrations of total phenols, flavonoids, and Glycyrrhizic acid. Using Response Surface Methodology (RSM) and Box-Behnken design, optimal extraction parameters were identified, showing that a balanced combination of ultrasonic power, argon-to-air ratio, and extraction time maximizes extract quality.

Ultrasonic treatment disrupted cell walls, increasing extract yields, while cold plasma, a non-thermal technique, effectively preserved heat-sensitive compounds. This combined method reduced the need for high temperatures and harmful solvents, making it a more sustainable and environmentally friendly alternative for extracting high-quality Liquorice root extracts. It holds promise for industrial-scale applications, offering a more efficient and eco-friendly approach to natural product extraction.

However, further research is needed to optimize parameters such as power intensity, treatment time, and environmental conditions. A thorough evaluation of the environmental and economic feasibility of scaling this method for industrial use is essential. Future studies should focus on optimizing extraction parameters for larger-scale applications, assessing the long-term stability and bioavailability of the extracted compounds, and exploring alternative plasma gases to improve extraction efficiency. For the application of this technology at an industrial scale, optimization of these parameters, along with a detailed investigation into the economic and environmental impacts, will be necessary. Once these challenges are addressed and conditions are optimized, this technology could become a more sustainable and environmentally friendly alternative for the industrial extraction of natural products.