3.5.1. Antioxidant Assays

#### Total Phenolic Content (TPC) by Spectrophotometric Assay

The TPC was determined using the Folin–Ciocâlteu method described by Mocan et al. [37]. For a high throughput of samples, a SPECTROstar Nano Multi—Detection Microplate Reader with 96-well plates (BMG Labtech, Ortenberg, Germany) was used. Briefly, a mixture solution consisting of 20 μL of extract, 100 μL of Folin-Ciocâlteu reagent, and 80 μL of sodium carbonate (Na2CO3, 7.5% *w*/*v*) was homogenized and incubated at room temperature in the dark for 30 min. Afterwards, the absorbance of the samples was measured at 760 nm. Gallic acid was used as a reference standard, and the TPC was expressed as gallic acid equivalents (GAE) in mg/g dry weight (d.w.) of plant material.

#### Total Flavonoid Content (TFC) by Spectrophotometric Assay

The total flavonoid content (TFC) was calculated and expressed as quercetin equivalents using a method previously described by Mocan et al. [38]. Briefly, a 100 μL aliquot of 2% AlCl3 aqueous solution was mixed with 100 μL of sample. After an incubation time of 15 min, the absorbance of the sample was measured at 420 nm. Quercetin was used as a reference standard, and the TFC was expressed as quercetin equivalents (QE) in mg/g dry weight (d.w.) of plant material.

## DPPH Radical Scavenging Assay

The capacity to scavenge the "stable" free radical DPPH, monitored according to the method described by Martins et al. [39], with some modifications, was performed by using a SPECTROstar Nano microplate reader (BMG Labtech, Offenburg, Germany). The reaction mixture in each of the 96-wells consisted of 30 μL of sample solution (in an appropriated dilution) and a 0.004% methanolic solution of DPPH. The mixture was further incubated for 30 min in the dark, and the reduction of the DPPH radical was determined at 515 nm. Trolox was used as a standard reference and the results were expressed as Trolox equivalents per g of dry weight herbal extract (mg TE/g d.w. of herbal extract).

#### Trolox Equivalent Antioxidant Capacity (TEAC) Assay

In the Trolox equivalent antioxidant capacity (TEAC) assay, the antioxidant capacity is reflected in the ability of the *Galium* extracts to decrease the color, reacting directly with the ABTS radical. The latter was obtained by oxidation of ABTS (2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid)) with potassium peroxydisulfate (K2S2O8). The amount of ABTS radical consumed by the tested

compound was measured at 760 nm after 6 min of reaction time. The evaluation of the antioxidant capacity was obtained using the total change in absorbance at this wavelength. The percentage of ABTS consumption was transformed in Trolox equivalents (TE) using a calibration curve.
