The freeze-dried RPTE was dissolved in distilled water to achieve a concentration of 50 mg/mL, and then filtered through a 0.45 μm membrane. Subsequently, the solution was applied onto a Sephadex G-25 gel filtration column (1.6 × 50 cm) (Ruji Technology Development Co., Ltd., Shanghai, China), which had been pre-equilibrated with distilled water [27 (link)]. Elution was carried out using distilled water at a flow rate of 2 mL/min, while monitoring the eluate at 280 nm with a UV-spectrophotometer (Huxi Analysis Instrument Factory Co., Shanghai, China). Fractions were collected and subsequently lyophilized.
The free radical scavenging activities of these fractions against DPPH•, hydroxyl radical (HO•), and superoxide anion radical (O2•−) were assessed individually [28 (link)]. The fraction exhibiting the most potent antioxidative activities was subjected to further purification using a Sephadex G-15 gel filtration column (2.6 × 60 cm, Ruji Technology Development Co., Shanghai, China). Elution was performed with distilled water at a flow rate of 1.25 mL/min. The fractions (RPTE2-1–RPTE2-4) were combined and lyophilized. Based on the same free radical scavenging assays, the fraction demonstrating the strongest antioxidant activity was selected for additional purification steps.
The free radical scavenging activities of these fractions against DPPH•, hydroxyl radical (HO•), and superoxide anion radical (O2•−) were assessed individually [28 (link)]. The fraction exhibiting the most potent antioxidative activities was subjected to further purification using a Sephadex G-15 gel filtration column (2.6 × 60 cm, Ruji Technology Development Co., Shanghai, China). Elution was performed with distilled water at a flow rate of 1.25 mL/min. The fractions (RPTE2-1–RPTE2-4) were combined and lyophilized. Based on the same free radical scavenging assays, the fraction demonstrating the strongest antioxidant activity was selected for additional purification steps.
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