The total phenolic content was determined by employing the methods given in the literature (Slinkard and Singleton, 1977 (link)) with some modification. Sample solution (1 mg/mL; 0.25 mL) was mixed with diluted Folin–Ciocalteu reagent (1 mL, 1:9, v/v) and shaken vigorously. After 3 min, Na2CO3 solution (0.75 mL, 1%) was added and the sample absorbance was read at 760 nm after a 2 h incubation at room temperature. The total phenolic content was expressed as milligrams of gallic acid equivalents (mg GAE/g extract) (Vlase et al., 2014 ).
The total flavonoids content was determined using AlCl3 method (Zengin et al., 2014 (link)). Briefly, sample solution (1 mg/mL; 1 mL) was mixed with the same volume of aluminum trichloride (2%) in methanol. Similarly, a blank was prepared by adding sample solution (1 mL) to methanol (1 mL) without AlCl3. The sample and blank absorbances were read at 415 nm after a 10 min incubation at room temperature. The absorbance of the blank was subtracted from that of the sample. Rutin was used as a reference standard and the total flavonoid content was expressed as milligrams of rutin equivalents (mg RE/g extract) (Mocan et al., 2015 (link)).
The total saponins content of the extract was determined by the vanillin-sulfuric acid method (Aktumsek et al., 2013 (link)). Sample solution (1 mg/mL; 0.25 mL) was mixed with vanillin (0.25 mL, 8%) and sulfuric acid (2 mL, 72%). The mixture was incubated for 10 min at 60°C. Then the mixture was cooled for another 15 min, followed by the sample absorbance measurement at 538 nm. The total saponin content was expressed as milligrams of quillaja equivalents (mg QAE/g extract).
The total triterpenoids content of the extracts was determined according to Zhang et al. (2010) (link) method with some modifications. Briefly, sample solution (1 mg/mL; 500 μL) was mixed with the vanillin–glacial acetic acid (5%, w/v, 0.5 mL) and 1 mL of perchloric acid. The mixture was incubated at 60°C for 10 min, cooled in an ice water bath for 15 min and then 5 mL glacial acetic acid was added and mixed well. After 6 min, the absorbance was read at 538 nm. Oleanolic acid was used as a reference standard and the content of total triterpenoids was expressed as oleanolic acid equivalents (mg OAE/g extract) through a calibration curve with oleanolic acid.
HPLC-PDA analyses were performed on a Waters liquid chromatograph equipped with a model 600 solvent pump and a 2996 photodiode array detector, and Empower v.2 Software (Waters Spa, Milford, MA, United States) was used for acquisition of data. A C18 reversed-phase packing column (Prodigy ODS (3), 4.6 × 150 mm, 5 μm; Phemomenex, Torrance, CA, United States) was used for the separation and the column was thermostated at 30 ± 1°C using a Jetstream2 Plus column oven. The injection volume was 20 μL. The mobile phase was directly on-line degassed by using Biotech DEGASi, mod. Compact (LabService, Anzola dell’Emilia, Italy). Gradient elution was performed using the mobile phase water-acetonitrile (93:7, v/v, 3% acetic acid) (Zengin et al., 2016 (link)). The UV/Vis acquisition wavelength was set in the range of 200–500 nm. The quantitative analyses were achieved at maximum wavelength for each compound.
The total flavonoids content was determined using AlCl3 method (Zengin et al., 2014 (link)). Briefly, sample solution (1 mg/mL; 1 mL) was mixed with the same volume of aluminum trichloride (2%) in methanol. Similarly, a blank was prepared by adding sample solution (1 mL) to methanol (1 mL) without AlCl3. The sample and blank absorbances were read at 415 nm after a 10 min incubation at room temperature. The absorbance of the blank was subtracted from that of the sample. Rutin was used as a reference standard and the total flavonoid content was expressed as milligrams of rutin equivalents (mg RE/g extract) (Mocan et al., 2015 (link)).
The total saponins content of the extract was determined by the vanillin-sulfuric acid method (Aktumsek et al., 2013 (link)). Sample solution (1 mg/mL; 0.25 mL) was mixed with vanillin (0.25 mL, 8%) and sulfuric acid (2 mL, 72%). The mixture was incubated for 10 min at 60°C. Then the mixture was cooled for another 15 min, followed by the sample absorbance measurement at 538 nm. The total saponin content was expressed as milligrams of quillaja equivalents (mg QAE/g extract).
The total triterpenoids content of the extracts was determined according to Zhang et al. (2010) (link) method with some modifications. Briefly, sample solution (1 mg/mL; 500 μL) was mixed with the vanillin–glacial acetic acid (5%, w/v, 0.5 mL) and 1 mL of perchloric acid. The mixture was incubated at 60°C for 10 min, cooled in an ice water bath for 15 min and then 5 mL glacial acetic acid was added and mixed well. After 6 min, the absorbance was read at 538 nm. Oleanolic acid was used as a reference standard and the content of total triterpenoids was expressed as oleanolic acid equivalents (mg OAE/g extract) through a calibration curve with oleanolic acid.
HPLC-PDA analyses were performed on a Waters liquid chromatograph equipped with a model 600 solvent pump and a 2996 photodiode array detector, and Empower v.2 Software (Waters Spa, Milford, MA, United States) was used for acquisition of data. A C18 reversed-phase packing column (Prodigy ODS (3), 4.6 × 150 mm, 5 μm; Phemomenex, Torrance, CA, United States) was used for the separation and the column was thermostated at 30 ± 1°C using a Jetstream2 Plus column oven. The injection volume was 20 μL. The mobile phase was directly on-line degassed by using Biotech DEGASi, mod. Compact (LabService, Anzola dell’Emilia, Italy). Gradient elution was performed using the mobile phase water-acetonitrile (93:7, v/v, 3% acetic acid) (Zengin et al., 2016 (link)). The UV/Vis acquisition wavelength was set in the range of 200–500 nm. The quantitative analyses were achieved at maximum wavelength for each compound.
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