| Sesaminol, an important lipid soluble sesame lignan, has so various physiological functions, such as antioxidation and anticarcinogenesis that it deserves a very broad application prospects. Sesaminol could be prepared by enzymatic hydrolysis of sesaminol triglucoside, the richest water-soluble lignan in sesame cake. Thus, the objective of this study was to optimize the HPLC method for the quantification of sesaminol triglucoside, extract STG from sesame cake, then the crude STG extract obtained was purified by procedures of polyamide column chromatography. Further, the study was conducted to produce sesaminol through hydrolysis of STG using the enzymes of β-glucosidase and cellulase, and the optimal conditions for this conversion were exploited, which provided theoretical foundation for industrial production of sesaminol.Firstly, the objective compound extracted was confirmed as STG by LC-MS(tR=24.71 min, m/z 855 [M-H]-). The HPLC method was optimized and validated for the quantification of STG. The chromatographic conditions were optimized as follows: Waters1525 HPLC equipped with UV detector and a Phenomenex(C18, 4.6 mm × l50 mm, 5 μm), the detection wavelength was 290 nm. The eluents used were methanol and distilled water. The elution conditions were linear gradient system from 30% methanol to 60% methanol for 45 min and the flow rate was 1.0 m L/min. Column temperature was 30℃, and 20 μL samples were injected into the column. Methodology validation experiment shows that the linear range of the method was 20~400 μg/mL, the average recovery rate was 96.80%~98.09%, and the relative standard deviations(RSD) were all under 2.00%.Secondly, physicochemical properties of sesame cake were determined to understand the characteristics of raw material. Then the effects of five commonly reagents of deionized water, ethanol, acetone, ethyl acetate, methanol on STG extraction were compared with the STG content in DSC(μg/g DSC) as index, which indicated that ethanol solution was the best choice. Further, investigation of the effects of various factors on STG extraction indicated that the extraction conditions were 75%, 25°C, 1:20(g/m L), 10 h, 2 times for ethanol concentration, temperature, solid/solvent ratio, extraction time and extraction times, respectively. On this condition, the content of STG achieved was 44.37 mg/100 g DSC.Thirdly, the crude extract obtained was purified by procedures of polyamide column chromatography, with the purity of 86.53%, laying the foundation for further research.Lastly, determination of β-glucosidase and cellulase indicated their activities were 36 U/mL and 77085 U/m L, respectively. Changes of STG and its hydrolysis products at different time during hydrolysis were determined by LC-MS. The results indicated that the main hydrolysis products were SDG(tR=31.20 min, m/z 693 [M-H]-), SMG(tR=38.23 min, m/z 531 [M-H]-) and sesaminol(t R=47.07 min, m/z 369 [M-H]-). And reasonable system of enzyme catalysis was set up using orthogonal experiment to optimize the conditions of hydrolysis of STG using β-glucosidase and cellulose. Under the optimal conditions of total enzyme dosage 100 μL, the ratio of β-glucosidase and cellulase 10:90(v/v), reaction time 28 h, substrate concentration 7 mg/mL, reaction temperature 45°C, and reaction system p H 4.8, the yield of sesaminol was 48.91%. |
PDF Full Text Request