*3.3. Phytochemical Analyses*

All the phytochemical analyses of the extracts were performed in triplicate (*n* = 3). All the freeze-dried extracts were dissolved in ethanol, a stock concentration of 1 mg/mL was prepared and used in analyses. All standards used in this study were purchased from Sigma-Aldrich (Arklow, Co. Wicklow, Ireland).

#### 3.3.1. Total Phenolic Content (TPC) and Total Phlorotannin Content (TPhC) Analyses

TPC and TPhC were determined following the Folin–Ciocalteu reagen<sup>t</sup> method as described by Rajauria et al. [50]. A total of 100 μL of sample/standard was mixed with 2 mL of sodium carbonate solution (Na2CO3 solution, 2% *w/v*). Following 2 min extraction, 100 μL of Folin–Ciocalteu's solution (1 M) was added to all the mixtures and the reaction was incubated for 30 min at room temperature in dark conditions. The absorbance of the reactions was read at 720 nm in a spectrophotometer (UVmini-1240, Shimadzu, Kyoto, Japan). Distilled water (instead of extract or standard) along with other reagents was used as blank and gallic acid (>97.5% purity) and phloroglucinol (>99% purity) at concentrations of 25–300 mg/L were used as standards for TPC and TPhC, respectively. TPC results were expressed as mg GAE/g and TPhC as mg PGE/g.

#### 3.3.2. Total Flavonoid Content (TFC)

TFC was determined following the protocol described by Liu et al. [51] with slight modifications. Briefly, 250 μL of sample/standard was mixed with 1.475 mL of distilled water and 75 μL of sodium nitrite solution (NaNO2 solution 5% *w/v*) and the reaction was allowed to stand for 6 min. A total of 150 μL of aluminium chloride hexahydrate solution (AlCl3·6H2O solution 10% *w/v*) was added, mixed thoroughly and allowed to stand for 5 min. Following extraction, 0.5 mL of sodium hydroxide solution (NaOH solution 1 M) was added and the absorbance of the reactions was read at 510 nm in a spectrophotometer. Distilled water (instead of extract or standard) along with other reagents was used as blank and quercetin (>95% purity) was used as standard at concentrations of 30–150 mg/L. TFC results were expressed as mg QE/g.

#### 3.3.3. Total Sugar Content (TSC)

TSC was determined by the phenol–sulphuric acid method as described by [52] with minor modifications. Briefly, 100 μL of sample/standard was mixed with 100 μL of phenol solution (0.8% *w/v*), followed by 2 mL of concentrated sulphuric acid (H2SO4 95–98%). The mixtures were allowed to stand for 10 min at room temperature and later incubated at 30 ◦C in a water bath for 20 min. The absorbance of the reaction was read in a spectrophotometer at 490 nm. Distilled water was used as blank and D-glucose (>99.5% purity) as a standard at concentrations of 50–250 mg/L. TSC results were expressed as mg GlcE/g.

#### 3.3.4. Total Tannin Content (TTC)

TTC was analysed following the protocol as described by Liu et al. [51]. A total of 50 μL of sample was mixed with 1.5 mL of a methanolic solution of vanillin (4% *w/v*) (99% purity, Sigma-Aldrich, Arklow, Co. Wicklow, Ireland) and 750 μL of hydrochloric acid (HCl 37% *w/v*). The solutions were mixed thoroughly and incubated in dark conditions at room temperature for 20 min. The absorbance of the reaction was read in a spectrophotometer at 500 nm. Distilled water was used as blank and (+)-catechin hydrate (>98% purity) was used as standard at concentrations of 15–150 mg/L. TTC results were expressed as mg ChE/g.
