Comparative extraction and simple isolation improvement techniques of active constituents' momilactone A and B from rice husks of Oryza sativa by HPLC analysis and column chromatography.

This paper reports comparative extraction efficiencies and enhancement methods for natural herbicidal (growth inhibitors) compounds, momilactone A and B, respectively from the dried husks of Oryza sativa using different extraction techniques and different solvent systems. Four different extraction techniques viz. percolation, agitation with heat, sonication and soxhlet using five solvent systems as ethyl acetate, acetone, acetonitrile, methanol and methanol:water (8:2) were evaluated. In these studies, it was observed that maximum extract yield was obtained using in methanol and methanol/water mixture as extracting solvent by soxhlet technique although the content of total momilactones A and B was higher in the methanol/water mixture in comparison to other extractions. The successive and simple isolation enrichment technique for momilactones A and B were achieved by solid-matrix partitioning after the treatment of methanolic extract with charcoal and using ethyl acetate as extracting solvent for momilactones A and B. The quantitative analysis of the extraction and enrichment development protocol was validated by a simple, accurate, reproducible RP-HPLC-UV-VIS method using a binary gradient elution comprising of acetonitrile and water (70:30). The separation was achieved on a waters Spherisorb S10 ODS 2 column (250 × 4.6 mm, I.D., 10 µm) that achieved a greater degree of linearity within an overall concentration of extracts and momilactones A and B, 1 mg mL^-1 and higher degree of correlation (0.9928 ≤ r² ≤ 0.9936) for momilactones A and B. So far, comparative extraction of momilactones A and B and HPLC of these compounds has not been reported. Standards of momilactones A (1) and B (2) were isolated along with other two compounds as orizaterpenoid (3) and 7-ketostigmaterol (4) from ethyl acetate extract of rice hulls of O. sativa and checked purity by HPLC-PDA-MS and identification of these isolated compounds (1-4) by complete spectroscopic techniques as IR, ¹H NMR, ¹³C NMR, 2D NMR and HR-MS. The qualitative analysis of momilactone A and B separation technique by thin layer chromatography was also developed.