**4. Extraction, isolation and identification of the active components in rice bran**

Different conventional methods are used to extract the bioactive compounds from plant materials. Of these, solvent, supercritical fluid, microwave‐assisted and ultrasonic‐assisted extraction are notable. Microwave‐assisted extraction and ultrasound‐assisted methods are used to detect antioxidant and anticancer bioactive components in the plant extracts [35, 36]. Imsanguan et al. described conventional solvent extraction, with different modifications at different temperatures (32–60°C), using 100 ml hexane at a rotating speed of 200 rpm for 24 h to extract γ‐oryzanol from rice bran [37]. However, this conventional technique does not fully remove toxic solvent residues from the final product; for this reason, Herrero et al. used the prominent technique of supercritical fluid extraction, which offers better extraction and purification of bioactive compounds [38]. Zigoneanu et al. described antioxidant extraction from rice bran oil using microwave‐assisted extraction, which uses electromagnetic radiation in the range of 0.3–300 GHz [39].

We developed the DF method to identify active components in the rice bran. As already described, the DF was derived from the solid fraction and chromatographed onto a silica gel column. One fraction derived from the methanol eluate was further fractionated using an octadecylsilane (ODS) column. The active fraction was obtained from the methanol/water (20– 70%) eluate and separated by high‐performance liquid chromatography (HPLC) using an ODS column. The BP‐lowering activity of each fraction was examined using a single oral adminis‐ tration to male, 14‐week old SHRSPs; we found that gavage of a certain fraction at 40 mg/kg body weight decreased BP significantly 1, 2, 4 and 6 h after administration. The chemical structure of this fraction was determined using fast atom‐bombardmentmass spectrometry, as well as NMR analyses; we then identified adenosine as the active compound [4].

γ‐Oryzanol was initially acknowledged as a single component when it was extracted from rice bran oil. Subsequently, 10 fractions were isolated using reverse‐phase HPLC and their structures were determined using gas chromatography‐mass spectrometry. Cycloartenyl ferulate, 24‐methylenecycloartanyl ferulate and campesteryl ferulate were identified as the major components of γ‐oryzanol [40–42].

Phenolic compounds were identified in the rice bran using sequential fractionation and subfractionation using Sephadex LH‐20 chromatography with 40% acetone. The total phenolic content was highest in the subfraction portion and the major phenolic acid was identified as FA (178.3 μg/mg) using HPLC and liquid chromatography‐electrospray ionization‐tandem mass spectrometry analyses [43, 44]. The chemical structure of the active components of rice bran is illustrated in **Figure 1**.

**Figure 1.** Chemical structure of bioactive components of rice bran.
