## 3 Results and Discussion  
### Spent Coffee Ground Characterization  
Spent Coffee Ground was supplied with an initial high moisture content, which was derived due to the extraction process via espresso machine. It was important to remove the contained moisture prior to any analysis. Considering the preservation of phenolic compounds, which might be unstable at high temperatures [36], the freeze-drying process was selected as the most suitable method to remove the contained moisture. The determined moisture content of raw SCG was 62 \(\pm\) 2%. The dried SCG was subjected to sequential extractions with four solvents of decreasing polarity, namely, water, MeOH:H\({}_{2}\)O (1:1), acetone:H\({}_{2}\)O (1:1), and pure acetone. The former two consist of higher polarity extracts, and the latter two consist of lower polarity extracts. Yields of both total solids and total phenols were determined. The results are presented in Table 2. The extract yields, in terms of total solids, were 60 \(\pm\) 9 mg/g\({}_{\mathrm{dry\;SCG}}\), 17 \(\pm\) 3 mg/g\({}_{\mathrm{dry\;SCG}}\), 11.0 \(\pm\) 0.4 mg/g\({}_{\mathrm{dry\;SCG}}\), and 103 \(\pm\) 5 mg/g\({}_{\mathrm{dry\;SCG}}\), respectively. Therefore, the total extractable material amounted to 191 mg/g\({}_{\mathrm{dry\;SCG}}\), of which 40% consisted of higher polarity compounds and 60% of lower polarity compounds. This is reasonable since SCG is the by-product of coffee brewed by hot water, and the majority of polar compounds have already been recovered. In contrast, only a minor part of the lipid fraction, mainly aroma compounds, has been extracted by hot water extraction, and the majority of lower polarity compounds are still present in SCG. As presented in Table 2, the Total Phenolic Content (TPC) of the extracts was also determined via the Folin-Ciocalteu method. The TPC yields of the extracts were also determined on the basis of dry SCG and amounted at 11.0 \(\pm\) 0.6 mg GAE/gdry SCG, 5.0 \(\pm\) 0.2 mg GAE/gdry SCG, 2.2 \(\pm\) 0.1 mg GAE/gdry SCG, and 3.3 \(\pm\) 0.1 mg GAE/gdry SCG, respectively. The Total Phenolic Content of the four extracts combined was 21.6 \(\pm\) 0.7 mg GAE/gdry SCG, whereas the contribution of higher and lower polarity extracts was 74% and 26%, respectively. The majority of phenolic compounds were extracted via the most polar solvent, that is, H\({}_{2}\)O (51.1%). MeOH:H\({}_{2}\)O (1:1) solvent extracted 23.3% of total TPC. Acetone:H\({}_{2}\)O (1:1) extracted 10.4%, while pure acetone extracted 15.2% of TPC. It is highly unlikely that the latter two successive extracts actually contain phenolic compounds but rather other reducing components that also respond to the Folin-Ciocalteu assay. The numerous non-phenolic compounds have been studied for interference with Folin-Ciocalteu method, as reported in the literature [37; 38; 39]. Among the compounds studied, several coffee-related compounds can be detected. The compounds in the respective literature that might be present in the lower polarity extracts of SCG are caffeine, theobromine, theophylline, furfural, and hydroxymethyl furfural, but none of the above have demonstrated interfering activity with the Folin-Ciocalteu reagent. However, the lower polarity ingredients of SCG, such as coffee oil, aroma compounds, coffee diterpenes, etc., constitute a complex mixture of components. Just the volatile fraction of coffee is composed of more than 900 compounds, which can be classified into several categories, such as (in order of abundance) furans, pyrazines, ketones, and pyrroles [40]. It would be very intriguing to search for non-phenolic, interfering compounds using the Folin-Ciocalteu method in such a complex mixture. According to the HPLC analyses, mainly water extracted the CQAs. The HPLC chromatograms of water and MeOH:H\({}_{2}\)O (1:1) extracts, monitored at 320 nm, are presented in Figure 1. Most of the peaks produced chlorogenic-type spectra, while no flavonoids were detected.