*4.3. LC-QToF-MS Analysis*

Metabolic profiling of plant tissues was performed using an Agilent 1290 Infinity LC system equipped with a quaternary pump, diode array detector (DAD), degasser, temperature-controlled column (25 ◦C), and cooled auto-sampler compartment (4 ◦C) which was coupled to an Agilent 6530 quadrupole time-of-flight (QToF) mass spectrometer (MS) with an Agilent Dual Jet Stream ionization source (Agilent Technologies, Melbourne, Australia). Full-scan mass spectra were acquired over an *m*/*z* range of 100–1700 Da at a rate of two spectra/second in both positive and negative ion modes. Chromatographic separation was achieved using a reverse-phase C18 Poroshell column (2.1 × 100 mm, 2.7 μm particle size) (Agilent Technologies, Santa Clara, CA, USA) equipped with a C18 guard column (2.1 × 12.5 mm, 5 μm particle size) (Agilent Technologies, CA, USA) using a flow rate of 0.3 mL min−1. The column was equilibrated for 40 min prior to analysis. Separation was obtained with a gradient of solvent A, [water (Milli-Q, TKA-GenPure, Germany) + 0.1% formic acid (LC-MS grade, LiChropur®, 98–100%, Sigma-Aldrich, MO, USA)] and solvent B [95% HPLC-grade acetonitrile (RCI Labscan, Bangkok, Thailand) + 0.1% formic acid]. The solvent gradient was as follows: 5% B for 0.5 min increasing to 100% B over the next 16.5 min, then holding at 100% B for 23 min and returning to 5% B from 23.1 min to 29 min. The DAD monitored absorbance across a range of wavelengths from 210 to 635 nm. Injection volume was 10 μL for each sample. Nitrogen was used as the drying gas at 250 ◦C and a flow rate of 9 L min−1. Five biological replicates for each treatment were analyzed. Phytoestrogens (isoflavones and coumestans) identified in annual pasture legumes in the current study are summarized in Table 5.

#### *4.4. Extraction of Polyphenols*

Foliar and inflorescence samples (1 g) from each pasture legume were freeze-dried and ground manually to a fine powder (≈1–2 mm size) using a mortar and pestle. Samples were extracted as described previously [77] with minor modifications. Briefly, ground foliar tissue was defatted with *n*-hexane, and residues were extracted three times with 20 mL of solvent (acetone:water:acetic acid 70:29.5:0.5 *v*/*v*/*v*). The three aliquots were combined, and solvent was removed using a rotary evaporator. The residue was lyophilized and reconstituted in 50% aqueous methanol to a final concentration of 1 g mL−<sup>1</sup> for storage at −20 ◦C until required.

#### *4.5. Quantification of Total Polyphenol Content*

Total free phenolic content was determined as described previously [78] with minor alterations. Briefly, 125 μL of the extract was mixed with 125 μL of Folin–Ciocalteu reagen<sup>t</sup> and 500 μL of deionized water and incubated in the dark for 6 min. The solution was neutralized by adding 1.5 mL 7% aqueous sodium carbonate solution and further incubated in the dark for 90 min, after which absorbance was measured at 725 nm using a UV/Vis spectrophotometer (FLUOstar Omega, BMG Labtech, Offenburg, Germany) against a methanol control. Total phenolic content was expressed as mg 100−<sup>1</sup> g of gallic acid equivalent (GAE). The experiment was repeated twice with three technical replicates.

#### *4.6. Quantification of Total Proanthocyanidin Content (TPAC)*

TPAC was quantified using a vanillin assay as described previously [79,80]. Briefly, 0.2 mL of extract was added to 0.5 mL of 1% (*w*/*v*) vanillin in methanol and 0.5 mL of 25% sulfuric acid in methanol. The extract was thoroughly blended using a vortex mixer and placed in a water bath at 37 ◦C for 15 min. Absorbance was measured at room temperature at a wavelength of 500 nm. The total proanthocyanidin content was determined as mg 100−<sup>1</sup> g of (+)-catechin equivalent (CE). The experiment was performed with three technical replicates and the experiment was repeated twice.
