Response Surface Methodology: An Optimal Design for Maximising the Efficiency of Microwave-Assisted Extraction of Total Phenolic Compounds from Coriandrum sativum Leaves

: The optimization of total phenolic compounds (TPC) extraction yield and maximization of total antioxidant capacity (TAC) from coriander leaves were investigated using response surface methodology. The extraction of TPC was carried out using microwave-assisted extraction. A Box-Behnken design was used to study the effects of the three independent variables, solvent concentration (ethanol/water 20–80%), microwave power (100–500 watt) and irradiation time (30–150 s) on the response. A second-order polynomial model was used to predict the reaction. The regression analysis showed that about 99% of the variations could be explained by the models. The predicted values were 50.97 GAE/g dw and 5.75 mg GAE/g dw for TPC and TAC. The reaction surface analysis showed that the optimum extraction parameters that maximized the extraction of antioxidants yield were 52.62% ethanol, 452.12 watt and 150 s. Under optimal conditions, the experimental values for TPC and TAC were 49.63 ± 0.93 mg GAE/g dw and 5.55 ± 0.07 mg GAE/g dw, respectively. The experimental values are in agreement with the predicted values, indicating the suitability of the model used and the success of the response surface methodology in optimizing the extraction conditions.


Introduction
Polyphenols are bioactive compounds found abundantly in various plant-based foods [1,2].They exhibit diverse biological effects, including antioxidant, anti-inflammatory, anticarcinogenic, and cardioprotective properties; studies highlight also their potential in reducing the risk of chronic diseases like cardiovascular diseases, cancer, diabetes, and neurodegenerative disorders [3][4][5][6].Polyphenols also play a crucial role in food preservation due to their antioxidant and antimicrobial activities.When used in food preservation, polyphenols help to prevent oxidation and spoilage by inhibiting the growth of microorganisms.This can extend the shelf life of food products and maintain their quality for longer periods of time [7].
Coriander, also known as cilantro, is a versatile herb that has a distinctive taste and aroma.It is widely used in different cuisines around the world and can be used in various forms, including leaves and seeds [1].Cilantro leaves are often used as a garnish or ingredient in dishes like salsa, guacamole, and curries, while the seeds can be ground into a powder or used whole in dishes like pickles, sausages, and curries.In addition to its culinary uses, coriander has numerous health benefits that make it a valuable addition to any diet.Coriander leaves are a good source of antioxidants, specifically polyphenols, that can protect the body against damage caused by free radicals.According to a study conducted by Ashika et al. [8], coriander leaves contain vanillic, p-coumaric, cisferulic, and trans-ferulic acids, which are responsible for the antioxidant properties of the plant.Additionally, Scandar et al. [9] found that the leaves and stems of coriander are the most nutritious and beneficial parts of the plant.Nhut et al. [10] found that coriander leaves contain rutin, a type of flavonoid that has antioxidant, anti-inflammatory, and anti-cancer properties.The study also found that the total flavonoids content, the compound considered as one of the secondary plant metabolites, demonstrates the effectiveness of biological roles such as inhibiting plasma platelet aggregation, histamine release, and antiviral activity.
Extraction is the first and most important step in the recovery and purification of bioactive compounds from plant materials.Among the various novel and sophisticated extraction techniques that have been introduced and studied to improve efficiency, microwaveassisted extraction (MAE) has garnered significant attention in recent years for its efficient extraction of bioactive compounds from various plant sources [11,12].MAE offers advantages such as reduced extraction times, higher yields, and lower environmental impact compared to traditional methods like Soxhlet extraction and maceration [13].The success of MAE relies on factors like solvent choice, exposure time, temperature, and equipment specifications [14].Additionally, the unique heating mechanism of microwaves allows for quicker extraction processes and better preservation of thermolabile compounds compared to conventional heating methods [15,16].Furthermore, MAE has been recognized as an environmentally friendly technique that minimizes energy consumption and solvent usage, making it ideal for extracting bioactive compounds from agro-industrial waste [17].
Response surface methodology is a statistical and mathematical technique for modelling and optimizing process parameters [18].Response surface methodology is highly useful in various fields such as engineering design optimization, chemical and biological process optimization, and natural product extraction [19].It provides a systematic approach to relate the input variables to the responses and is a cost-effective and efficient method for optimizing experimental processes.The importance of response surface methodology lies in its ability to approximate the relationship between input and output variables, which supports the optimization of complex systems and reveals synergistic effects of process parameters.The methodology includes the design of experiments, the analysis of results and the creation of models to improve the results in different applications.The planes used in the response surface methodology study are quadratic planes such as the Box-Behnken planes or the central composite planes (Box-Wilson).Response modelling is performed using regression techniques that allow a response to be linked to a set of factors [20].
The aim of this study is to enhance the extraction efficiency of antioxidants from coriander leaves using microwave-assisted extraction.This will be achieved by modeling and optimizing the extraction conditions, which include solvent concentration, microwave power, and irradiation time.Response surface methodology will be utilized to determine the optimal conditions for extracting total phenolic compounds and total antioxidant capacity.

Sample Preparation
In this study, coriander plant (Coriander sativum) was procured from a local market located in the Bejaia region of Northern Algeria.Leaves were separated from the stem and washed with tap water followed by distilled water, and left to dry for approximately 48 h at room temperature in a ventilated and dark room.The dried sample was then ground to obtain a fine powder with a diameter of less than 250 µm using a grinder (A11 basic grinder from Ika, Staufen in Germany).The powder was stored at −20 • C before analysis.The moisture content was determined by constant weight at 105 • C and was found to be 5.0 ± 0.5%.The water activity (Aw) was measured using a Hygro Palm Aw instrument (Rotronic AG Bassersdorf, Switzerland) and was found to be 0.18 ± 0.2 at a temperature of 20.6 • C.

Experimental Work
To optimize the microwave-assisted extraction procedure, a series of single-factor experiments were conducted to determine the effects of individual process parameters.This approach was chosen to minimize the total experimental work required while still providing valuable insights for improving the procedure.Through this method, the impact of each parameter on the process could be assessed and the most significant factors could be identified.This allowed for a more targeted and efficient optimization process (Table 1).The variable was kept constant (50%, 500 watt and 150 s for ethanol concentration, microwave power and irradiation time, respectively) when it was not studied.In both the single-factor trials and the consecutive response surface methodology optimizations, the focus was on evaluating the extraction yield of total phenolic compounds and the maximization of total antioxidant capacity.
Following that, an optimization of the processes was carried out utilizing a response surface methodology approach based on a Box-Behnken design to refine the conditions (Table 2).The experimental design applied was a three-level three-factor Box-Behnken design, and the required number of experiments (N) was determined using the formula outlined in Equation ( 1 where k is the number of factors and C 0 is the number of central points (3).
To analyze the data, a regression analysis was conducted to fit a second-order polynomial equation (quadratic model) based on the general equation (Equation ( 2)) in order to predict the optimal conditions for the extraction process.
where y represents the response function; a 0 is a constant coefficient; a i , a ii and a ij are the coefficients of the linear, quadratic and interactive terms, respectively, and x i and x j represent the coded independent variables.The factor levels were coded as −1 (low), 0 (central point or middle) and 1 (high).The variables were coded according to the following equation (Equation ( 3)): where x i represents a new variable (dimensionless) encoded from the original variable X i .X 0 represents the reference value or initial value, and ∆X represents the increment or discretization step.
After conducting the analysis of variance, the regression coefficients for the individual linear, quadratic, and interaction terms were determined.To illustrate the impacts of independent variables and their interactions, three-dimensional surface plots were created from the fitted polynomial equation using the regression coefficients.
To confirm the reliability of the model, further extraction experiments were conducted at the predicted optimal conditions determined by the response surface methodology.The experimental results obtained were then compared to the values forecasted by the regression model.

Microwave-Assisted Extraction Process
For the optimization of the microwave-assisted extraction process, the following parameters influencing the extraction process were selected: solvent concentration, microwave power and irradiation time.A quantity (1 g) of coriander leaf powder was placed in a 250 mL bottom flask containing ethanol-water quantities.The suspension was extracted at different concentrations of solvent, microwave power and irradiation time.The extracts were separated by centrifugation at 3000 rpm (NF 200, Nüve, Turkey) for 10 min and stored at 4 • C until use.Fifteen tests were performed before the optimum was determined.An extraction was then performed under the optimum conditions determined using response surface methodology.The total phenolic compounds and total antioxidant capacity were considered in the response surface methodology optimization and model validation tests.

Response Parameters 2.5.1. Determination of the Total phenolic Compounds
The total phenolic compounds (TPC) of the plant samples was determined using the Folin-Ciocalteu reagent as reported by Singleton and Rossi [21].A volume of 0.1 mL of the extract was mixed with 0.8 mL Folin-Ciocalteu reagent (10% v/v) and 0.4 mL sodium carbonate (7.5% w/v).The absorbance was measured at 720 nm (UV/Vis spectrophotometer, Biotech Engineering Management Co., Ltd., Nicosia, Cyprus) after 60 min of incubation at room temperature against a blank (made as reported for the sample but with 0.1 mL of sample solvent).The results were expressed as milligrams of gallic acid equivalent per gram of dry weight (mg GAE/g dw) by referring to a calibration curve.

Evaluation of the Total Antioxidant Capacity
The total antioxidant capacity (TAC) of the plant samples was evaluated using the DPPH radical method as reported by Brand-William [22].A volume of 0.2 mL of the extract was mixed with 1 mL of methanolic DPPH solution (60 µM).The absorbance was measured at 515 nm after 30 min of incubation at room temperature against a blank (made as reported for the sample but with 0.2 mL of sample solvent).The results were expressed as milligrams of gallic acid equivalent per gram of dry weight (mg GAE/g dw) by referring to a calibration curve.

Statistical Analysis
All extraction trial and subsequent analyses were performed in triplicate, and the results are reported as means ± standard deviation (SD).The impact of each factor on the TPC yield and TAC in the single-factor experiment for microwave-assisted extraction was evaluated through ANOVA and Tukey's post hoc test at a 95% confidence level to determine their statistical significance.The data collected from the Box-Behnken design experiments for microwave-assisted extraction were subjected to ANOVA to assess the significance and appropriateness of the model for the response variable.A significance level of p < 0.05, p < 0.01 and p < 0.001 was considered as indicating significant, highly and very highly significant results, respectively.The JMP (Version 17.0, SAS, Cary, NC, USA) software was utilized to analyze all the experimental results and create the Box-Behnken design.

Effect of Single Factors
The influence of different extraction parameters on TPC yield and TAC was investigated using a method that varied one parameter at a time.The factors examined were ethanol concentration, microwave power, and irradiation time.
Choosing the right extraction solvent is crucial for determining the quantity and quality of the extracted phenolic compounds.Acetone, ethanol, and methanol are widely used solvents for extracting phenolic compounds from plants [23]; nevertheless, given the toxicity concerns associated with methanol and acetone, which are not suitable for food applications, ethanol solvent was chosen instead.Ethanol offers several advantages over other solvents, including higher extraction efficiency, environmental friendliness, and lower cost.The efficiency of extraction is significantly influenced by the concentration of ethanol, as reported in the literature [24].Therefore, the impact of varying ethanol concentration was evaluated to optimize the extraction efficiency.Based on Table 1, the value of TPC and TAC ranged from 37.85 ± 0.24 to 42.91 ± 0.48 mg GAE/g dw and 2.92 ± 0.11 to 3.91 ± 0.21 mg GAE/g dw, respectively.As can be seen, the TPC and TAC increased significantly from the concentration of 10 to 50%, and then decreased to achieve a value of 38.65 ± 0.59 mg GAE/g dw and 2.89 ± 0.04 mg GAE/g dw, respectively.A similar tendency was reported for the extraction of total polyphenols from other plant material [25][26][27].The effect of ethanol concentration on the extraction of antioxidants can be attributed to polarity changes.As the ethanol concentration in the solvent increases, its polarity decreases, which enhances the extraction of less polar components [28].Moreover, the increase in ethanol concentration also promotes the breakdown of cell membranes, which in turn enhances the solvent's ability to penetrate the solid matrix during the extraction process [29,30].At higher ethanol concentrations, the resulting polarity becomes unsuitable for extracting antioxidants from coriander leaves, making it less effective for this purpose.Given these findings, the concentration range of 20-80% was chosen for the response surface methodology trials.
The selection of microwave power is crucial in determining the efficiency and yield of the phenolic compounds extraction process.Higher microwave power, which leads to the increase in the temperature, can accelerate the extraction by disrupting hydrogen bonds, enhancing solvent penetration into the matrix, and facilitating the release of target compounds [31]; however, it is important to note that there is a limit to this effect and beyond a certain point, increasing the microwave power may not lead to any further improvements in the extraction yield as higher microwave power levels can induce degradation of phenolic compounds due to elevated temperatures [32].As shown in Table 1, the value of TPC yield and TAC ranged from 35.96 ± 0.47 to 40.97 ± 0.93 mg GAE/g dw and 2.98 ± 0.09 to 3.89 ± 0.08 mg GAE/g dw, respectively.The variation of microwave power over the range of 100-900 watt caused the increment in TPC yield and TAC which was achieved at 300 watt followed by a significant decrease.Our result was lower than those reported in the literature [33].Based on these results, the microwave power range 100-500 watt was selected for the response surface methodology trials.
According to the Table 1, the value of TPC and TAC ranged from 38.66 ± 0.14 to 43.08 ± 0.27 mg GAE/g dw and 2.92 ± 0.04 to 4.03 ± 0.07 mg GAE/g dw, respectively.As can be seen, the TPC and TAC increased significantly from the irradiation time of 30 to 90 s, and then decreased to achieve the value of 38.88 ± 0.71 mg GAE/g dw and 2.92 ± 0.04 mg GAE/g dw, respectively, after an extended extraction time.Our result was higher than that reported in the literature [12].Based on these results, the irradiation time range 30-150 s was selected for the response surface methodology trials.

Construction of the Experimental Plan
The optimization of the antioxidants extraction from coriander leaves by response surface methodology is operated using the Box-Behnken model based on the maximization of TPC extraction yield and TAC as response variables.Three parameters (independent variables), solvent concentration (ethanol 20-80%), microwave power (100-500 watt) and irradiation time (30-150 s) were investigated.The ranges (the lower and upper ends) of each independent variable were determined based on the result of the single factor effect from preliminary work (Section 2.2).The factors levels, observed and predicted values of TPC and TAC were summarized in Table 3.The amount of TPC and the maximization of TAC from Coriandrum sativum leaf extract using the MAE method ranged from 26.34 to 49.70 mg GAE/g dw and 3.08 to 5.75 mg GAE/g dw, respectively.The highest yield in TPC and TAC was observed under extraction conditions of 50% (v/v) solvent concentration, 500 watt microwave power, and an irradiation time of 150 s.Hihat et al. [1], who investigated the effect of oven and microwave drying on the total polyphenols and antioxidant capacity of coriander leaves, reported values of 48.44 mg GAE/g dw and 82.21%, respectively.

Analysis of the Model
To assess the significance of the model, an ANOVA analysis was conducted.Table 4 shows the ANOVA results for the effects of solvent concentration, microwave power and irradiation time on TPC and TAC, relative to the dry weight of coriander leaves.The coefficients of determination (R 2 ) for the TPC and TAC models are 0.994 and 0.984, respectively, indicating that only a very small percentage (0.06 and 0.16%, respectively) of the total variation remains unexplained by these models.In addition, the adjusted coefficients of determination (R 2 adj) for the TPC and TAC models are 0.982 and 0.956, respectively, indicating close agreement between the experimental and predicted values.The lack of fit test is used to determine if the model is appropriate for describing the experimental data or if another model should be selected.The lack of fit test values for TPC and TAC are 0.2441 and 0.1695, respectively, which are higher than 0.05 and not significant compared to the pure error.This suggests that the current model is sufficient to fit the experimental data.
The regression coefficients for the intercept, the linear, quadratic and interaction terms of the models were calculated using the least squares method and presented in Table 5.The ANOVA of regression coefficient showed a linear response based on the p-value of solvent concentrations (x 1 ), which were highly significant (p < 0.0001), followed by microwave power (x 2 ) and irradiation time (x 3 ) for both TPC and TAC.In the interaction between variables, only solvent concentration-microwave power (x 1 x 2 ) had a significant effect on TPC (p < 0.0051) and TAC (p < 0.0038).While in the quadratic model, only solvent concentrations (x 1 2 ) and microwave power (x 3 2 ) were significant for TPC (p < 0.0001 and p < 0.0002, respectively), and solvent concentration (x 1 2 ) and irradiation time (x 3 2 ) for TAC (p < 0.0001 and p < 0.0314, respectively).The mathematical equations correlating the TPC and TAC (Equation (4) and Equation (5), respectively) with process variables are given below in terms of coded factors excluding non-significant terms.

Analysis of Plots Describing Factors Effect
The most effective method for illustrating the impact of an independent variable on the yield of TPC extraction and TAC is to generate response surface plots using a model.This involves manipulating two variables within the experimental range being studied, while holding the remaining variable at its central level (0 level) [34].Figure 1 demonstrates the influence of solvent concentration, microwave power, and irradiation time on the yield of TPC extraction and TAC from coriander leaves.
As can be seen in Figure 1a, the increase in solvent concentration (x 1 ) and microwave power (x 2 ) resulted in an augmented yield of TPC and TAC.However, higher solvent concentration (x 1 ) and microwave power (x 2 ) had a negative effect on the extraction, probably due to the decrease in polarity of the solvent [35] and/or the degradation of antioxidants [36].Moreover, analysis of the surface plots showed that there were optimal levels of concentration of TPC and TAC, which is due to the quadratic effect of the solvent concentration (x 1 2 ) as corroborated in Table 5.Additionally, the impact of solvent concentration was observed to be statistically significant in interaction with microwave power (x 1 x 2 ) (p value 0.0051 and 0.0038 for TPC and TAC, respectively).This finding was in agreement with the study which reported the significance of ethanol percentage on the yield extraction of total polyphenols from Pistacia lentiscus [33].Polarity plays a crucial role in extracting antioxidants from various plant and marine sources.Several studies have shown that the polarity of the solvent affects the antioxidant activity of the extracts.By increasing the ethanol concentration in the solvent, the cell membrane breaks down, allowing the solvent to more easily penetrate the solid matrix [29].Moreover, increasing the ethanol concentration in the solvent reduces its polarity, which enhances the extraction of less polar components [28].However, using a very high ethanol concentration is not suitable for extracting TPC from plant material.According to Figure 1b, the yield of TPC and TAC is affected by the increase in solvent concentration (x 1 ) and irradiation time (x 3 ).It was observed that both factors independently influenced the extraction of TPC and TAC.Additionally, analyzing the surface plots revealed that there were optimal concentration levels for TPC and TAC, which is attributed to the quadratic effect of irradiation time (x 3 2 ) as supported by Table 5.The impact of solvent concentration in interaction with irradiation time (x 1 x 3 ) was found to be not significant (p value 0.5754 and 0.2713 for TPC and TAC, respectively).An elongated extraction time allows for prolonged contact between the plant material and the solvent, facilitating the transfer of phenolic compounds into the solvent.This can lead to increased extraction yields as more compounds have the opportunity to be released from the plant matrix [37].
Based on the Figure 1c, increasing microwave power (x 2 ) and irradiation time (x 3 ) resulted in higher yields of TPC and TAC.In other words, both microwave power (x 2 ) and irradiation time (x 3 ) simultaneously influenced TPC and TAC.However, it was observed that microwave power had a greater impact on TPC extraction than irradiation time.It was reported that the higher absorption of microwave energy led to increased temperatures within the sample, causing cell rupture and facilitating the release of antioxidant compounds [38]; however, excessive power may also degrade sensitive compounds [39].Further, many studies have reported a decline in the levels of recovered TPC when using microwave-assisted extraction for long periods.Zhang et al. [40] observed that the yields of phenolic compounds of A. blazei increased significantly from 1 to 5 min during MAE, followed by a slight decrease.

Validation of the Model
To assess the predictive power of the model, the optimal conditions were determined based on maximum desirability.The optimal conditions for achieving the highest TPC and maximum TAC were found to be 52.62%ethanol concentration, 452.12 watt microwave power, and 150 s irradiation time.Under these conditions, the experimental values for TPC and TAC were 49.63 ± 0.93 mg GAE/g dw and 5.55 ± 0.07 mg GAE/g dw, respectively.These experimental results were in good agreement with the predicted values for TPC and TAC, which were 50.97 and 5.75 mg GAE/g dw, respectively.Zeković et al. [12] investigated coriander seeds and reported optimal microwave-assisted extraction conditions with an ethanol concentration of 63%, an extraction time of 19 min, and an irradiation power of 570 watt to simultaneously maximize total polyphenols yield and increase antioxidant activity.Their predicted values for TPC and antioxidant activity (IC 50 ) were 311.23 mg GAE/100 g and 0.0315 mg/mL, respectively.

Conclusions
The response surface methodology was employed to explore the individual and interactive effects of three variables, namely solvent concentration, microwave power and irradiation time, with the objective of optimizing the microwave-assisted extraction of TPC and the maximization of TAC from Coriander sativum.The high correlation of the mathematical model indicates that a quadratic polynomial model can be used for modelling the solid-liquid extraction of TPC and TAC.From the response surface plots, all three investigated factors significantly affected the TPC extraction yield and the maximization of TAC.The experimental values (49.63 ± 0.93 mg GAE/g dw and 5.55 ± 0.07 mg GAE/g dw for TPC and TAC, respectively) agreed with the predicted values (50.97 GAE/g dw and 5.75 mg GAE/g dw for TPC and TAC, respectively) and clearly showed the suitability of the developed quadratic models.These results confirm the predictability of the model for TPC from coriander leaves and TAC under the experimental conditions used (52.62% aqueous ethanol as solvent, 452.12 watt as microwave power and 150 s as irradiation time).This optimized process, which is simple, fast, efficient and non-denaturing, can be used for the extraction of substances of interest in both research and industrial settings.

Figure 1 .
Figure 1.Effects of solvent concentration and microwave power (a), solvent concentration and irradiation time (b), and microwave power and irradiation time (c) on TPC (mg GAE/g dw) and TAC (mg GAE/g dw).

Figure 1 .
Figure 1.Effects of solvent concentration and microwave power (a), solvent concentration and irradiation time (b), and microwave power and irradiation time (c) on TPC (mg GAE/g dw) and TAC (mg GAE/g dw).

Table 1 .
Results of single-factor experiments for microwave-assisted extraction from coriander leaves.

Table 2 .
Independent variables affecting the microwave-assisted extraction.

Table 3 .
Box-Behnken design matrix, experimental and predicted values of total phenolic compounds (TPC) and total antioxidant capacity (TAC).

Table 4 .
Analysis of model variance and lack of fit for total phenolic compounds (TPC) and total antioxidant capacity (TAC) of coriander leaves.

Table 5 .
Regression coefficient, standard error, and Student's t-test results of response surface of TPC and TAC referred to dry weight of coriander leaves.