**Metabolic Profiling Provides Unique Insights to Accumulation and Biosynthesis of Key Secondary Metabolites in Annual Pasture Legumes of Mediterranean Origin**

#### **Sajid Latif 1,2,\*, Paul A. Weston 1,3, Russell A. Barrow 1,4, Saliya Gurusinghe 1, John W. Piltz 5 and Leslie A. Weston 1,3**


Received: 27 May 2020; Accepted: 19 June 2020; Published: 28 June 2020

**Abstract:** Annual legumes from the Mediterranean region are receiving attention in Australia as alternatives to traditional pasture species. The current study employed novel metabolic profiling approaches to quantify key secondary metabolites including phytoestrogens to better understand their biosynthetic regulation in a range of field-grown annual pasture legumes. In addition, total polyphenol and proanthocyanidins were quantified using Folin–Ciocalteu and vanillin assays, respectively. Metabolic profiling coupled with biochemical assay results demonstrated marked di fferences in the abundance of coumestans, flavonoids, polyphenols, and proanthocyanidins in annual pasture legume species. Genetically related pasture legumes segregated similarly from a chemotaxonomic perspective. A strong and positive association was observed between the concentration of phytoestrogens and upregulation of the flavonoid biosynthetic pathway in annual pasture legumes. Our findings sugges<sup>t</sup> that evolutionary di fferences in metabolic dynamics and biosynthetic regulation of secondary metabolites have logically occurred over time in various species of annual pasture legumes resulting in enhanced plant defense.

**Keywords:** pasture legumes; phytoestrogens; flavonoids; coumestans; polyphenols; proanthocyanidins; metabolic profiling; biosynthesis
