**2. Results**

#### *2.1. Quantification of Phytoestrogens*

Concentration of coumestans in foliar tissues of all species ranged between 0.13 and 48.4 mg kg−<sup>1</sup> and varied significantly across pasture species (Table 1). Both coumestrol and 4-methoxycoumestrol accumulated at higher concentrations in leaf and stem compared to inflorescence tissues. Bladder clover possessed significantly higher concentrations of both coumestrol and 4-methoxycoumestrol in leaf tissue compared to other pasture legumes (48.4 and 24.8 mg kg−1, respectively) while in stem tissue, bladder clover had the highest concentration of coumestrol (39.6 mg kg−1). Lucerne contained the highest concentration of 4-methoxycoumestrol (27.7 mg kg−1) in any tissue of all species, and lucerne inflorescence tissue contained the highest concentrations of coumestrol and 4-methoxycoumestrol (0.5 and 0.2 mg kg−1, respectively) of all annual pasture species examined.


**Table 1.** The concentration of coumestrol and 4-methoxycoumestrol (mg kg−1) at physiological maturity in selected annual pasture legume species.

ND denotes compounds not detected. Least significant difference (LSD) differentiated between concentration means. Values represent the arithmetic mean of five replicates ± standard deviation. \*cv. is acronym for cultivar.

Three isoflavones—daidzein, formononetin and genistein—are commonly found in pasture legumes and were also profiled in this study (Table 2). Isoflavone content in leaf (113.3–443.9 mg kg−1), stem (37.5–968.1 mg kg−1), and inflorescence (29.8–614.1 mg kg−1) varied significantly by both species and tissue (Table 2). Gland clover had the highest concentration of total isoflavones in leaf tissue (443.9 mg kg−1) while bladder clover had the highest concentration in stem and inflorescence tissue

(968.1 and 604.1 mg kg−1, respectively). In terms of individual phytoestrogenic isoflavones in aerial tissues, formononetin concentration was greatest overall, followed by genistein at physiological maturity (Table 2). The production of phytoestrogenic isoflavones was greatest in clover species; specifically, daidzein concentration was highest in leaf tissues of gland clover at 120.2 mg kg−1. Production of these compounds in stems was greatest in bladder clover and subterranean clover (112.0 and 107.8 mg kg−1, respectively) when compared to other species. Subterranean clover contained the highest levels of daidzein (128.9 mg kg−1) in inflorescence tissue when compared to other annual pasture species.


**Table 2.** The concentration of daidzein, formononetin and genistein (mg kg−1) in annual pasture legume species at physiological maturity, post-flowering.

Least significant difference (LSD) was used to differentiate between concentration means. Values represent the arithmetic mean of five replicates ± standard deviations.

Formononetin concentration in leaf tissue was significantly higher in the perennial legume lucerne (329.4 mg kg−1) than the other species, while for stem tissue, formononetin concentration was greatest in bladder clover (829.8 mg kg−1). Interestingly, genistein levels were greatest in all three tissues types in bladder clover compared to other species.

#### *2.2. E*ff*ect of Biserrula Cultivar and Growth Stage on Phytoestrogen Levels*

Given the strong potential of biserrula to produce large quantities of biomass and suppress weeds successfully over time, further evaluation was performed to examine temporal effects on the accumulation of phytoestrogens in aerial tissues in both of the commercially available cultivars of biserrula in Australia. Analysis of tissues of Casbah and Mauro analyzed at five different growth stages showed that total concentrations of the phytoestrogens differed significantly between cultivars (Figure 2a,b). Phytoestrogen concentrations reached their maxima at either 50% bloom or full bloom

while the lowest concentrations were observed at crop senescence. Significant differences in the concentration of coumestans between the two cultivars were limited to coumestrol at pre-bloom and 50% bloom stages; cv. Mauro was observed to produce greater levels of coumestans than cv. Casbah. The phytoestrogenic isoflavones formononetin and daidzein were more abundant in tissues of Casbah than Mauro, while the opposite was true for genistein.

**Figure 2.** (**a**) The concentration of coumestrol and 4-methoxycoumestrol at five growth stages i.e., pre bud, pre bloom, 50% bloom, full bloom, and senescence of field-grown biserrula cv. Casbah and cv. Mauro averaged over two years. Error bars indicate standard deviation; (**b**) the concentration of selected isoflavones at five growth stages i.e., pre bud, pre bloom, 50% bloom, full bloom, and senescence in biserrula cv. Casbah and cv. Mauro samples averaged over two years. Error bars indicate standard deviation.

#### *2.3. Quantification of Total Polyphenols and Proanthocyanidins*

Extractable TPC (total polyphenol content) ranged from 4.40 to 13.84 GAE and TPAC (total proanthocyanidins) ranged from 1.73 to 6.49 mg 100−<sup>1</sup> g CE (Table 3). Gland clover contained significantly higher extractable TPC levels (13.84 mg 100−<sup>1</sup> g) compared to all other pasture species. Extractable and bound TPAC was significantly higher in biserrula cv. Casbah compared to other species, while bound TPAC was only detected in the two biserrula cultivars (Casbah and Mauro) and the perennial legume, lucerne.



Values represent arithmetic mean of five replicates. Values followed by the same superscript in each column are not significantly different (*p* < 0.05) as determined by ANOVA.

#### *2.4. Abundance of Flavonoids and Their Glycosides*

The clustering of molecular features profiled through non-targeted metabolic profiling revealed that molecular entities varied between species but were similar in species of the same genera (Supplementary Materials Figure S1). Over 5000 molecular features in total were successfully profiled in legume leaf tissues, with 1727 in stem and 1503 in inflorescence tissues (Figure S2). Interestingly, flavonoids and their glycosides accounted for the majority of constituents among all annotated major classes of secondary metabolites, based on verification with analytical standards as well as METLIN database comparisons. The relative abundance of various flavonoids and their glycosides in selected pasture legumes is presented in Figure 3. Interestingly, the total number of molecular features characterized in this study was highest in biserrula followed by French and yellow serradella, while gland clover exhibited the fewest total number of molecular features (Supplementary Materials Figure S1). However, gland clover, a recent introduction to Australia obtained from native pastures in the Mediterranean, exhibited a considerably higher abundance of secondary metabolites including flavonoids and their glycosides profiled in this study compared to other species (Figure 3). Both Casbah and Mauro cultivars of biserrula presented chemically similar profiles and exhibited relatively low abundance of flavonoids and their glycosides, in contrast to subterranean and gland clover.

**Figure 3.** Hierarchal clustering of relative abundance of flavonoids, their glycosides, and coumestrol in leaf tissue in pasture legumes collected in 2016. Hierarchical clustering algorithm and Euclidean distance metric were used on normalized abundance using Mass Profiler Professional MPP (ver. 14.5 Agilent Santa Clara, CA, USA).
