3.2. Oleoresin Composition
In parallel with the extraction efficiency results, it was not surprising to observe that the solvent type had significant (P< 0.05) effect on the total lipid, protein and phenolic content of oleoresins. Following the traditional solvent extraction approach, the highest protein (0.67 ± 0.03 wt%), lipid (69.22 ± 0.04 wt%) and phenolic contents (48 ± 0.01 mg GAE/g) were obtained with IL1 (Table 1). The lowest extraction for protein (0.36 ± 0.02 wt%) was obtained using dichloromethane, while the lowest lipid (53.12 ± 0.08 wt%) and phenolic contents (21 ± 0.02 mg GAE/g) were observed using ethanol. Wet milling extraction with a ball mill increased (P < 0.05) the total lipid (82.19±0.07 wt%), protein (1.37 ± 0.05 wt%), and phenolic content (57 ± 0.01 mg GAE/g) of oleoresin when IL1 used as compared to total lipid ( 69.22 ±0.04 wt%), protein 0.67 ± 0.03 wt%), and phenolic content (48 ± 0.01 mg GAE/g) with traditional extraction (Table 1). In a similar way to traditional extraction, IL1 and IL2 were same (P> 0.05) for lipid, protein, phenolic content. Jin et. al (2022) also found that using ball mill increased the overall content of protein, fat, and volatile compounds in the sesame seed extracts as compared to colloid and stone mills. The increase was higher for protein (51.1%) than lipid (15.8 %) at the conditions for the highest extraction yield when IL is used (Table 1).
The fatty acid composition of the oleoresins was analyzed by using gas chromatography and shown in Table 2. Oleic acid, palmitic acid, palmitoleic acid and myristic acid were the most abundant fatty acids in all the oleoresins. Octadecanoic acid was present only in trace amounts for all extracts with a concentration range between 0.292 to 1.792%. This aligned with the fatty acid content of raw sorghum varieties of reported in previous studies (Espitia-Hernández et al., 2022). The solvent type had a significant (P < 0.05) effect on the fatty acid composition. The unsaturated fatty acids (oleic and palmitoleic acids) increased more than saturated ones (myristic and palmitic acids) when ILs were used instead of dichloromethane and ethanol. This was probably related to the double bonds of unsaturated fatty acids that provided lower free energy for temporal ionization and to interact with cations and anions of ionic liquids. In another study, Li et al. (2009) showed that higher polyunsaturated fatty acid extracts from cod liver oil were obtained with ILs in comparison to organic solvents, such as acetone, methanol, acetonitrile, and chloroform. The percentages of oleic, palmitic and myristic acids were not significantly (P > 0.05 ) different when IL1 and IL2 were used with traditional extraction process while it was significantly (P < 0.05 ) different for palmitoleic acid. In contrast, the percentage of these fatty acids were significantly (P < 0.05 ) different when dichloromethane or ethanol was used. The concentrations of both oleic and palmitic acids were significantly (P < 0.05 ) lower with both traditional solvents than ILs when traditional extraction was conducted.
The extraction method also had a significant (P < 0.05 ) effect on the fatty acid composition (Table 2). The percentage of oleic and palmitoleic acids significantly (P < 0.05 ) increased while myristic and palmitic acids significantly (P > 0.05 ) decreased when ball milling process was conducted instead of traditional process. The highest concentration of oleic acid, the most abundant unsaturated fatty acid in the oleoresins was 23.663% ± 0.003 with the combined action of ball milling and IL1. In addition to this, the increase in oleic acid was 45.89% and 29.15% when ball milling was used with IL2and ethanol respectively, as compared to traditional solvent extraction with the same solvents. On the other hand, the decrease in myristic acid was 24.57% and 34.44% when ball milling was used with IL2 and ethanol respectively, as compared to traditional extraction using IL2 and ethanol. Similar changes in unsaturated (palmitoleic and oleic acids) and saturated (myristic and palmitic acids) fatty acid concentrations were observed by Korber et. al (2022) where ball mill extraction was applied for lettuce plants (Lactuca sativa L.) to extract galactolipids and sulfolipids. Furthermore, the percentages of oleic, palmitoleic, myristic and palmitic acids were not significantly (P > 0.05 ) different when IL1 and IL2 were used with ball milling extraction. Similar to the traditional extraction, the percentage of these fatty acids changed when dichloromethane and ethanol were selected. The use of ILs increased (P < 0.05 ) the total phenolic content of the oleoresins. Oleoresins were rich in eight phenolic compounds: caffeic acids, coumaric acid, ferulic acids, apigenidin, luteolidin, 7-methoxy apigeninidin, eriodictyol and narencenin (Table 3). The HPLC chromatogram was given in Supplementary Materials (Fig. S1). Caffeic acid, coumaric acid and luteolidin were the major phenolic compounds present in all oleosome samples. Among these, caffeic acid followed by coumaric acid were the most abundant and they remained so with all types of solvents. The solvent type significantly (P < 0.05) affected the total phenolic content and composition (Table 3). The percent increase in caffeic acid was 74.84% when IL1 was used instead of dichloromethane with the traditional extraction process. In addition to this, there was no significant (P > 0.05) difference between IL1 and IL2 while ethanol and dichloromethane showed significant (P < 0.05) difference using the same extraction method.
The extraction method also had a significant (P < 0.05 ) effect on the phenolic composition (Table 3). The increase in caffeic acid was 28.93% and 50.16% when ball milling was combined with IL1 and dichloromethane respectively, as compared with the traditional extraction process using the same solvents. Similar effects were observed in luteolinidin when ball milling and traditional extraction processes were compared: the increase in luteolinidin was 21.78% and 57.68% when ball milling extraction was performed with IL2 and ethanol respectively, as compared with traditional extraction. Furthermore, the percentages of caffeic acid were not significantly (P > 0.05 ) different when IL1 and IL2 with the combined action of ball milling process, but there was a slight significant difference when ethanol and dichloromethane were used. Similarly, in another study, Talekar et al. (2019) showed that total phenolics and antioxidant “punicalagin” increased when ball-mill pre-treatment was used for pomegranate peel waste, and it was more efficient than ultrasound-assisted and Soxhlet extraction.