# Optimized Isolation Procedure for the Extraction of Bioactive Compounds from Spent Coffee Grounds
## 3 Results and Discussion  
Due to the importance of the compounds contained in SCGs, a response surface methodology was employed to finely tune the parameters that affect their extraction from SCGs and obtain extracts rich in these compounds that can be used for the production of beverages or the enrichment of other products. This is not the first study to examine the extraction of compounds from SGCs. [PERSON] et al. [15] focused only on the extraction of caffeine and chlorogenic acid from SCGs using accelerated solvent extraction. Similarly, [PERSON] et al. [25] optimized the extraction of caffeine, trigonelline, and nicotinic acid from SCGs. However, the parameters that they evaluated were the solvent extraction volume and the extraction temperature. [PERSON] et al. [26] focused on the extraction of polyphenols from SGCs using three different techniques. However, for the conventional extraction method, the effect of extraction time was not studied. Our aim was to examine all important parameters for the extraction of caffeine, caffeic acid, chlorogenic acid, and neochlorogenic acid, as well as total polyphenols. As such, four extraction parameters were examined in a wide range of values. The parameters that were examined were (\(X_{1}\)), the solvent (water and ethanol were examined, as well as their mixtures), (\(X_{2}\)), the extraction time, ranging from 18 to 222 min, (\(X_{3}\)), the temperature (ranging between 20 and 80 \({}^{\circ}\)C), and (\(X_{4}\)), the ratio of liquid to solid (ranging between 5 and 35 mL/g). In order to optimize the extraction of the compounds as well as examine the effect of each extraction factor, an RSM approach was employed. The responses were the caffeine content of the extracts, the TPC, as well as their FRAP and DPPH values. The content of caffeic acid, chlorogenic acid, and neochlorogenic acid was also examined in each extract. Their concentrations in the extracts followed the same trend as the TPC. Based on ANOVA and summary-of-fit tests, the adequacy of the response surface and model fitting was evaluated, considering how closely the measured and predicted values corresponded. The experimental conditions of the prepared extracts, as well as their responses, are reported in Table 2. Moreover, Table 2 includes data regarding the responses of the extract prepared using the lower examined levels (\(-2\)) for each parameter, for means of comparison. Table 3 shows also statistical information about the models used to describe the relationship between the independent variables and the response variables. This information includes the second-order polynomial equations, the coefficients of each term in the equations, and other statistical parameters that quantify the goodness of fit of the models to the data. It was discovered that all of the coefficients increased, indicating that the models fit the data well. The desirability function was used to determine the maximum predicted values for caffeine concentration, TPC, and antioxidants (FRAP and DPPH), as well as the optimal levels for each of the four variables taken into consideration (Figure S1-S4, respectively). Additionally, bivariate response fitting plots are shown in Figures S5-S7, and Figures 1-4 provide 3D response graphs for each response. As regards the caffeine content of the extracts, the ethanol ratio of the solvent (\(X_{1}\)) and the ratio of liquid to solid (\(X_{4}\)) were statistically highly significant, as well as the interactions \(X_{1}\times X_{3}\) and \(X_{1}\times X_{4}\). In Figure 1, it can be seen that caffeine was more readily extracted when water was used. The increase in ethanol content in the solvent resulted in a decreased extraction of the compound. This can be justified by the hydrophilic nature of caffeine, due to which it can form hydrogen bonds with water molecules, while the number of hydrogen bonds formed with ethanol would be less [27]. Moreover, it can be seen that in order to increase the extraction yield, an increased liquid-to-solid ratio was needed.