Agilent hplc vials

Extracts were prepared in methanol and water (1:1, v/v). Briefly, 1 g of powdered sample was mixed with 20 mL of methanol–water mixture. The mixture was placed on a water bath at 30 °C for one hour. Then, the samples were centrifuged at 4000 rpm for 10 min. After that, 2 mL from each sample was filtered using Agilent membrane polytetrafluorethylene (PFTE) (Agilent Technologies, Waldbronn, Germany) with pore size of 0.45 µm and transferred into 2 mL Agilent HPLC vials. Similarly, two more extracts in 100% methanolic and 100% pure deionized water were also prepared using similar procedures. One part of the above extracts were used for the determination of total phenolic contents (TPC) using Folin–Ciocaltaeu Reagent. The results of TPC for both methods of analyses were compared.

Stored samples were freeze-dried until constant weight. Grains were de-husked manually for samples from 5 DAF, while for samples from 10 DAF onwards, a customised manual de-husker was used to loosen the husk from whole grain and further husk removal was performed manually. Samples were ground to powder using a laboratory ball mill (Mixer ill MM301, Retsch).
A subsample of ~50 mg finely ground grain was extracted in a 15 mL falcon tube with 10 mL of methanol acidified with 1N HCl (85:15, v/v) for anthocyanin extraction and separately with 1 mL absolute methanol for γ-oryzanol extraction. The mixture was sonicated for 30 min in a Soniclean Ultrasonic cleaner (100% maximum power) at room temperature. The crude extract was centrifuged at 12,000 g for 10 min at room temperature. The resultant supernatant (~800 μl) was pipetted into 2 mL Agilent HPLC vials and stored at -20°C until analysis using High-Performance Liquid Chromatography (HPLC).