TITLE: Isolation of the Antioxidant Metabolite from the EAF
AUTHORS: Jing Xu

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>EAF (4.13 g), which showed the most antioxidantactivity, was subjected to column chromatography on a silica gel column chromatography (CC), employing a step gradient of CH</span><span style = "vertical-align:sub;">2</span><span>Cl</span><span style = "vertical-align:sub;">2</span><span>-CH</span><span style = "vertical-align:sub;">3</span><span>OH (10:1, 10:2, 10:3, 10:5, 1:1, 0:1, v/v) and afforded eleven fractions (Fr. 1-Fr. 9) (Fig. 1). Fr. 2 was subjected to open silica gel CC using a successive elution of EtOAc-CH</span><span style = "vertical-align:sub;">3</span><span>OH (10:1-0:1, v/v) to yield fractions Fr. 2.1-2.4. Fr. 2.2 and 2.4 were isolated using Sephadex LH-20 CC/ODS-HPLC to afford </span><span style = "vertical-align:sub;">6</span><span> (8.1mg, 0.20%) and</span><span style = "vertical-align:sub;"> 7 </span><span>(20mg, 0.48%). Fr. 4 was subjected to polyamide CC using CH</span><span style = "vertical-align:sub;">2</span><span>Cl</span><span style = "vertical-align:sub;">2</span><span>-CH</span><span style = "vertical-align:sub;">3</span><span>OH-HCOOH as eluent (10:2:1, v/v). Promising subfraction Fr. 4-3 was eluted using CH</span><span style = "vertical-align:sub;">3</span><span>OH-H</span><span style = "vertical-align:sub;">2</span><span>O (1:1-1:0, v/v) withRP C-18 CC. Final purification was achieved by polyamide CC using CH</span><span style = "font-weight:bold;">2</span><span>Cl</span><span style = "font-weight:bold;">2</span><span>-CH</span><span style = "font-weight:bold;">3</span><span>OH (10:4, v/v) to yield </span><span style = "vertical-align:sub;">1</span><span> (0.803 g, 19.44%).Fr.5 was subjected to polyamide CC with CH</span><span style = "font-weight:bold;">2</span><span>Cl</span><span style = "font-weight:bold;">2</span><span>-EtOAc-CH</span><span style = "font-weight:bold;">3</span><span>OH(5:5:1,v/v) as the solvent. Fr.5-3 and Fr. 5-5 were isolated using polyamide/RP C-18/Sephadex LH-20 CC to yield</span><span style = "vertical-align:sub;"> 5</span><span> (11mg, 0.27%), </span><span style = "vertical-align:sub;">11 </span><span>(8mg, 0.19%) and </span><span style = "vertical-align:sub;">12 </span><span>(5mg, 0.12%). Fr.7.1 collected from Fr. 7 with EtOAc-CH</span><span style = "font-weight:bold;">3</span><span>OH(10:2, v/v) were passed through polyamide CC, and purified by ODS-HPLC using a gradient of CH</span><span style = "vertical-align:sub;">3</span><span>OH-H</span><span style = "font-weight:bold;">2</span><span>O(3:7-9:1,v/v))as eluent to yield </span><span style = "vertical-align:sub;">3</span><span> (10mg, 0.24%) and </span><span style = "vertical-align:sub;">9</span><span> (5mg, 0.12%). Fr. 8-1, Fr.8-3 and Fr.8-4 obtained from Fr. 8 with CH</span><span style = "font-weight:bold;">2</span><span>Cl</span><span style = "font-weight:bold;">2</span><span>-CH</span><span style = "font-weight:bold;">3</span><span>OH(10:2,v/v) were isolated using ODS- HPLC/Sephadex LH-20 CC to yield </span><span style = "vertical-align:sub;">2 </span><span>(150mg, 3.63%), </span><span style = "vertical-align:sub;">4 </span><span>(5mg, 0.12%), </span><span style = "vertical-align:sub;">8</span><span> (3mg, 0.07%) and </span><span style = "vertical-align:sub;">10</span><span> (8mg, 0.19%).EAF (4.13 g), which showed the most antioxidantactivity, was subjected to column chromatography on a silica gel column chromatography (CC), employing a step gradient of CH</span><span style = "font-weight:bold;">2</span><span>Cl</span><span style = "font-weight:bold;">2</span><span>-CH</span><span style = "font-weight:bold;">3</span><span>OH (10:1, 10:2, 10:3, 10:5, 1:1, 0:1, v/v) and afforded eleven fractions (Fr. 1-Fr. 9) (Fig. 1). Fr. 2 was subjected to open silica gel CC using a successive elution of EtOAc-CH</span><span style = "font-weight:bold;">3</span><span>OH (10:1-0:1, v/v) to yield fractions Fr. 2.1-2.4. Fr. 2.2 and 2.4 were isolated using Sephadex LH-20 CC/ODS-HPLC to afford </span><span style = "vertical-align:sub;">6</span><span> (8.1mg, 0.54‱) and</span><span style = "vertical-align:sub;"> 7 </span><span>(20mg, 1.33‱). Fr. 4 was subjected to polyamide CC using CH</span><span style = "font-weight:bold;">2</span><span>Cl</span><span style = "font-weight:bold;">2</span><span>-CH</span><span style = "font-weight:bold;">3</span><span>OH-HCOOH as eluent (10:2:1, v/v). Promising subfraction Fr. 4-3 was eluted using CH</span><span style = "font-weight:bold;">3</span><span>OH-H</span><span style = "font-weight:bold;">2</span><span>O (1:1-1:0, v/v) withRP C-18 CC. Final purification was achieved by polyamide CC using CH</span><span style = "font-weight:bold;">2</span><span>Cl</span><span style = "font-weight:bold;">2</span><span>-CH</span><span style = "font-weight:bold;">3</span><span>OH (10:4, v/v) to yield </span><span style = "vertical-align:sub;">1</span><span> (0.803 g, 0.54%).Fr.5 was subjected to polyamide CC with CH</span><span style = "vertical-align:sub;">2</span><span>Cl</span><span style = "vertical-align:sub;">2</span><span>-EtOAc-CH</span><span style = "vertical-align:sub;">3</span><span>OH(5:5:1,v/v) as the solvent. Fr.5-3 and Fr. 5-5 were isolated using polyamide/RP C-18/Sephadex LH-20 CC to yield</span><span style = "vertical-align:sub;"> 5</span><span> (11 mg, 0.73‱), </span><span style = "vertical-align:sub;">11 </span><span>(8 mg, 0.53‱) and </span><span style = "vertical-align:sub;">12 </span><span>(5 mg, 0.33‱). Fr.7.1 collected from Fr. 7 with EtOAc-CH</span><span style = "vertical-align:sub;">3</span><span>OH(10:2, v/v) were passed through polyamide CC, and purified by ODS-HPLC using a gradient of CH</span><span style = "vertical-align:sub;">3</span><span>OH-H</span><span style = "vertical-align:sub;">2</span><span>O(3:7-9:1,v/v))as eluent to yield </span><span style = "vertical-align:sub;">3</span><span> (10 mg, 0.66 ‱) and </span><span style = "vertical-align:sub;">9</span><span> (5 mg, 0.33‱). Fr. 8-1, Fr.8-3 and Fr.8-4 obtained from Fr. 8 with CH</span><span style = "vertical-align:sub;">2</span><span>Cl</span><span style = "vertical-align:sub;">2</span><span>-CH</span><span style = "vertical-align:sub;">3</span><span>OH(10:2,v/v) were isolated using ODS- HPLC/Sephadex LH-20 CC to yield </span><span style = "vertical-align:sub;">2 </span><span>(150 mg, 0.1%), </span><span style = "vertical-align:sub;">4 </span><span>(5 mg, 0.33‱), </span><span style = "vertical-align:sub;">8</span><span> (3 mg, 0. 20‱) and </span><span style = "font-weight:bold;">10</span><span> (8 mg, 0.53‱).</span></div></div>

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