**2. Extraction and purification**

The most popular method used for extraction of isoflavonoids is maceration with either MeOH or EtOH containing various percentages of H2 O at room temperature followed by liquid-liquid fractionation using solvents with different polarities [6, 10, 19–32]. Another method of extraction used MeOH or EtOH under reflux or in soxhlet apparatus [5, 33–36]. Mixture of MeOH and CHCl3 or CH2 Cl2 (1:1) was also applied for extraction [37–41]. Other research groups extracted the plant materials with acetone [42–44], CHCl3 [45, 46], CH2 Cl2 [47–50] or diethyl ether [51] at room temperature. Successive extraction starting with petroleum ether or hexane, CHCl3 , EtOAc and MeOH using soxhelt apparatus [52–56] was also reported. The isoflavone contents of soybeans were extract using supercritical fluid extraction [57].

The majority of purification and isolation steps utilized silica gel in the form of column, Preparative Thin Layer Chromatography (PTLC) or Centrifugal Preparative Thin Layer Chromatography (CPTLC) [19, 21, 45]. Combination of silica gel and Sephadex LH-20 was also applied for isoflavonoid purification [6, 10, 54, 55]. In addition to silica gel, semi-preparative C<sup>18</sup> High Performance Liquid Chromatography (HPLC) columns were used for final purification of isoflavonoids [23, 30, 31, 38, 48]. The polar *n*-butanol fraction of *Ononis serrata* was fractionated on C18 silica gel applying the Vacuum Liquid Chromatography (VLC) technique followed by normal silica gel column for purification of isoflavonoid glucosides [27]. Two isoflavenes were isolated from *Lespedeza homoloba* after chromatography on porous polymer gel Diaion followed by silica gel column. Final purification step was performed on preparative C18 HPLC column [36]. Isoflavonoids from *Iris germanica* were purified by silica gel VLC and CC, and final purification was achieved via LiChrolut EN/RP-18 solid phase extraction tubes [26]. High-speed counter-current chromatography (HSCCC) was applied for the purification of flavan glycoside and isoflavones from *Astragalus membranaceus*, the seeds of *Millettia pachycarpa* and soy flour [20, 58, 59]. Isolation and identification of isoflavanones, biflavanones and bisdihydrocoumarins were achieved using Liquid Chromatography- Mass Spectrometry (LC-MS), Liquid Chromatography- Solid Phase Extraction- Nuclear Magnetic Resonance (LC-SPE-NMR) and Electronic Circular Dichroism (ECD). In this method, MS of target compounds was measured directly in the LC effluent. For NMR analyses, the peaks were collected from 20 LC runs, loaded on SPE cartilages, dried with nitrogen gas and finally eluted with CD3 OD [32].
