# Optimized Isolation Procedure for the Extraction of Bioactive Compounds from Spent Coffee Grounds
## 3 Results and Discussion
Plot **(A)**, covariation of \(X_{1}\) (C, %) and \(X_{2}\) (\(t\), min); plot **(B)**, covariation of \(X_{1}\) (\(C\), %) and \(X_{3}\) (\(T\), \({}^{\circ}\)C); plot **(C)**, covariation of \(X_{1}\) (\(C\), %) and \(X_{4}\) (\(R\), mL/g); plot **(D)**, covariation of \(X_{2}\) (\(t\), min) and \(X_{3}\) (\(T\), \({}^{\circ}\)C); plot **(E)**, covariation of \(X_{2}\) (\(t\), min) and \(X_{4}\) (\(R\), mL/g); plot **(F)**, covariation of \(X_{3}\) (\(T\), \({}^{\circ}\)C) and \(X_{4}\) (\(R\), mL/g). As regards the TPC content of the extracts, the effects of all the examined parameters (\(X_{1}\), \(X_{2}\), \(X_{3}\) and \(X_{4}\)) were found to be statistically significant, as well as the quadratic terms \(X_{1}{}^{2}\) and \(X_{2}{}^{2}\). However, the effect of temperature on the extraction, as well as that of the liquid-to-solid ratio were less pronounced, compared to those of the two other factors. As can be seen in Figure 2, the extraction of TPC increased as the ethanol content of the solvent increased up to 50% \(v/v\). A further increase in the ethanol content resulted in a decreased extraction of the polyphenols. This can be attributed to the change in the polarity of the solvent, as polyphenols are more readily extracted in less polar solvents than in water [15]. The maximum predicted value for TPC was 19.85 mg GAE/g of dry SCGs, which could be achieved by adding 35 mL of a 50% \(v/v\) water/ethanol solution to 1 g of SCGs and stirring the mixture for 120 min at 65 \({}^{\circ}\)C. The results obtained are comparable to those previous studies [26; 28]. As regards neochlorogenic acid, chlorogenic acid, and caffeic acid, they followed the same trend as the TPC. [PERSON] et al. [25] used a microwave-assisted extraction (MAE) method to extract TPC from SCGs. They found that the maximum TPC content was obtained at a solvent ratio of 50% ethanol and 50% water, an extraction time of 30 min, and a temperature of 80 \({}^{\circ}\)C, yielding 19.62 mg GAE/g. The optimal extraction conditions were different from those used in the current study, but the results were comparable in terms of the maximum predicted TPC content (19.85 mg GAE/g) obtained in the current study. As regards the ferric-reducing antioxidant power of the extracts, the effects of all the examined parameters (\(X_{1}\), \(X_{2}\), \(X_{3}\), and \(X_{4}\)) were found to be statistically significant, as well as those of the quadratic terms \(X_{1}{}^{2}\) and \(X_{2}{}^{2}\). The FRAP response was affected in a similar way as the TPC. This was somewhat expected, since the antioxidant properties of the extracts, stemmed, mainly, from the polyphenols. This was also the case with the DPPH response. The maximum response in the FRAP assay can be achieved by adding 35 mL of a 50% \(v/v\) water/ethanol solution to 1 g of SCGs and stirring the mixture for 120 min at 80 \({}^{\circ}\)C. Likewise, the maximum response in the DPPH assay was achieved by adding 35 mL of a 50% \(v/v\) water/ethanol solution to 1 g of SCGs and stirring the mixture for 222 min at 50 \({}^{\circ}\)C.  
As can be seen in Table 4, the optimum extraction parameters for each response varied. However, according to the desirability plots (Figures S1-S4), the temperature variations did not cause significant changes in the predicted response. As such, in order to render the extraction method more environmentally friendly and lower its overall cost, 20 \({}^{\circ}\)C is suggested as the optimum extraction temperature for the TPC, FRAP, and DPPH responses. In order to examine whether the predicted values could be achieved in the proposed conditions, two additional extractions were conducted. SCGs were extracted using the optimum conditions for caffeine extraction, and another extract was prepared using the conditions for TPC. In both cases, the extraction was carried out at 20 \({}^{\circ}\)C. According to the results, the caffeine content of the extract was found to be 6.6 mg/g of dry SCGs.