Oleanolic Acid

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 optimized chromatographic analysis was carried out using a Waters Alliance 2695 liquid chromatography system (Waters, Milford, CT, USA) equipped with 2996 photodiode array detector (Waters, Milford, CT, USA) and ACE C18 (150 × 4.6 mm, 3 μm) column (ACT, Aberdeen, UK). Since all triterpenoids cannot be separated in a single chromatographic run, different chromatographic conditions were used.
For the analysis of triterpenoid acids (maslinic, corosolic, betulinic, oleanolic, ursolic acids) and neutral triterpenoids with chromophores (betulin, erythrodiol, uvaol), the mobile phase consisted of acetonitrile and water (89:11, v/v), delivered at a flow rate of 0.7 mL/min in the isocratic mode. The column temperature was set at 20 °C with an injection volume of 10 μL. Whereas the isocratic elution system for the analysis of neutral triterpenoids, which lacks chromophores (lupeol, β-amyrin, α-amyrin, friedelin) and phytosterol (β-sitosterol), consisted of acetonitrile and methanol (10:90, v/v). The column temperature was set at 35 °C, the flow rate was 1 mL/min, and the sample injection volume was 10 μL.
Detection of all triterpenoids was performed at a wavelength of 205 nm corresponding to the maximum absorption, and peaks were identified with retention times as compared with standards.

Standards of the following phenolic compounds were obtained from PhytoLab GmbH & Co. KG (Vestenbergsgreuth, Germany): apigenin, apigenin-7-O-glucoside, trans-cinnamic acid, p-coumaric acid, trans-ferulic acid, protocatechuic acid, maslinic acid, vanillic acid, tyrosol, hydroxytyrosol, hydroxybenzoic acid, hydroxytyrosol acetat, oleanolic acid, oleoside-11-methylester, luteolin, luteolin-7-O-glucoside, (+)-pinoresinol, 1-acetoxypinoresinol, oleuropein, oleacein, p-HPEA-EA, and oleocanthal. Acetonitrile, methanol, water, and formic acid (LC-MS grade) were purchased from Sigma Aldrich Chemie GmbH (Buchs, Switzerland). 2 M Folin-Ciocalteau reagent and anhydrous sodium carbonate (both from Sigma) were used to measure the TPC.

The total phenol content was determined using the Folin–Ciocalteau method, according to Colonna et al. [13 (link)], with some modifications. The extracts were prepared using 0.1000 g of dry, raw material and 10 mL of 80% methanol. In a test tube, 0.6 mL of extract, 3 mL of Folin–Ciocalteu reagent, and 2.0 mL of Na₂CO₃ 7.5% were added. After the mixture was vortexed, it was allowed to react for 60 min in the dark. Finally, the absorbance at 765 nm was measured with a spectrophotometer (UV-2600, SHIMADZU, Tokyo, Japan) using distilled water as a blank. The content of total phenols was expressed as equivalent mg of gallic acid per g of the dry sample.
Total flavonoid content was estimated by the AlCl₃ colorimetric method [47 ]. Briefly, 20 mg/mL of the extract was prepared using 60% ethanol. The extract of 2 mL was used with 0.4 mL of a 10% (p/v) aluminum trichloride (AlCl₃) solution and 0.4 mL of 5% sodium nitrate (NaNO₂). Subsequently, the mixture was vortexed and incubated for 6 min. Then, 4 mL of NaOH (4%) was added to stop the reaction. Finally, the absorbance at 510 nm was measured with a spectrophotometer (UV-2600, SHIMADZU) using 60% ethanol as a blank. The total flavonoid content was expressed as equivalent mg of rutin per g of the dry sample.
Total saponins (TSs) were quantified by the colorimetric method according to Le et al. [17 (link)], with some modifications. The extracts were prepared using 0.1000 g of dried plant tissue and 7 mL of 70% (v/v) ethanol, and sonicated at 55 °C for 40 min. In a test tube, evaporate 0.2 mL of the extract in a water bath at a temperature of 70 °C and add 0.1 mL of 5% vanillin reagent and 0.4 mL of perchloric acid. The reaction was heated in a water bath at 60 °C for 15 min, and ethyl acetate (4 mL) was added for 10 min. Finally, the absorbance at 560 nm was measured with a spectrophotometer (UV-2600, SHIMADZU, Tokyo, Japan) using 70% ethanol as a blank. The total saponin content was expressed as equivalent mg of oleanolic acid per g of the dry sample.

All solvents, reagents, and standards were of analytical reagent grade. Acetone, methanol, acetonitrile, hydrochloric acid, 4-(Dimethylamino) cinnamaldehyde and reference standarts for β-Amyrin, maslinic acid, oleanolic acid, corosolic acid, ursolic acid, α-Amyrin, quercetin-3-rhamnoside, myricetin, quercetin-3-α-L-arabinopyranoside and quercetin-3-α-L-arabinofuranoside were purchased from Sigma-Aldrich (Steinheim, Germany). Ethanol 96% (v/v) was purchased from AB Stumbras (Kaunas, Lithuania), formic acid was bought from Merck (Darmstadt, Germany), and Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) was purchased from Scharlau (Sentmenat, Spain). Reference standards for myricetin-3-galactoside, cyanidin chloride, peonidin chloride, malvidin-3-galactoside, malvidin-3-arabinoside, malvidin chloride, peonidin-3-arabinoside, cyaniding-3-galactoside, delphinidin-3-galactoside, peonidin-3-glucoside, peonidin-3-galactoside, cyaniding-3-glucoside and cyaniding-3-arabinoside were purchased from Extrasynthese (Genay, France). Standards for quercetin and quercetin-3-galactoside were purchased from Carl Roth (Karlsruhe, Germany). The standard for quercetin-3-O-glucoside was bought from Biochemistry (Buchs, Switzerland). Purified deionized water was provided using a water purification system (Milli-Q^®, Millipore, Bedford, MA, USA).

The test of PLA2 inhibitory activity was performed as discussed before by De Arajo and Radvanybm [59 (link)]. Commercially available phospholipase A2 procured from Sigma-Aldrich (Saint Louis, MO, USA) (P6534), was used in this assay. In 100 mL H₂O, the substrate was made up of 3.5 mM lecithin, 3 mM NaTDC, 100 mM NaCl, 10 mM CaCl₂, and 0.055 mM red phenol as a colorimetric indicator. Phosphate buffer was used to adjust the pH of the reaction mixture to 7.6. The sPLA2 protein was solubilized in 10% acetonitrile at a concentration of 0.01 to 0.08 g/L. For 20 min at room temperature, a volume of 10 L of these PLA2 solutions was incubated with a volume of 10 L containing 10 g of each compound. After that, 1 mL of PLA2 substrate was added, and the hydrolysis kinetics was monitored for 5 min by monitoring the optical density at 558 nm. The percentage of inhibition was estimated by comparing the results to a control experiment (devoid of compound). Oleanolic acid was used as a positive control in this experiment.