Isolation and Structure Determination of 24-Methylenecycloartanyl Ferulate from Indian Rice Bran and Its Quantitative Analysis

BACKGROUND: γ-oryzanol is a major bioactive constituent in rice. Most of the literature reports isolation of 24-methylenecycloartanyl ferulate (24-mCAF) from rice bran oil (RBO) of other than Indian variety. Current research has successfully applied high-performance thin layer chromatography (HPTLC) method for isolation of 24-mCAF from Indian variety (Indrayani) of RBO. MATERIALS AND METHODS: HPTLC method was developed for standard γ-oryzanol using tinidazole as an internal standard. The proposed HPTLC method was optimized and validated as per the guidelines stated by the International Conference on Harmonization Q2 R1 recommendations. The mobile phase composed of toluene:ethyl acetate:methanol (15.0:1.7:3.3, (v/v/v) was selected because well-resolved peaks were obtained. The optimum wavelength chosen for detection and quantitation was 317 nm. RESULTS: The retention factors for tinidazole, 24-mCAF, and CAF were found to be 0.27 ± 0.02, 0.72 ± 0.02, and 0.79 ± 0.02, respectively. The percent content of 24-mCAF in ethanol fraction was found to be 1.02%. The 24-mCAF was isolated from RBO using HPTLC method. CONCLUSION: The characterization data of 1D, 2D spectral analysis confirm that the isolated compound 1 is 24-mCAF. SUMMARY: HPTLC method was developed for standard γ-oryzanol using tinidazole as an internal standard. The proposed HPTLC method was optimized and validated as per the guidelines stated by the ICH Q2 R1 recommendations. The characterization data of 1D, 2D spectral analysis confirms that the isolated compound is 24-methylenecycloartanyl ferulate. In this work, high purity 24-mCAF was successfully isolated from crude RBO using HPTLC with a solvent system composed toluene: ethyl acetate: methanol (15.0:1.7:3.3, v/v/v). Abbreviations used: RBO: Rice Bran Oil, CAF: Cycloartenol ferulic acid, 24-mCAF: 24-Methylcycloartenol ferulic acid, HPLC: High-Performance Liquid Chromatography, HPTLC: High-Performance Thin Layer Chromatography, 1H: Proton nuclear magnetic resonance spectroscopy, 13C: Carbon-13 nuclear magnetic resonance spectroscopy, COSY: Correlation spectroscopy, NOESY: Nuclear overhauser effect spectroscopy, HMBC: Heteronuclear multiple bond correlation nuclear magnetic resonance spectroscopy, HSQC: heteronuclear single quantum coherence nuclear magnetic resonance spectroscopy.