| Summary: | 碩士 === 中華醫事科技大學 === 醫學檢驗生物技術系碩士班 === 100 === Ferulic acid (4-hydroxy-3-methoxycinnamic acid) is an abundant phenolic acid that is present in the plant cell wall and has an important role in linkage of hemicellulosic polysaccharides with other cell wall components. Ferulic acid with several potential industrial and medical applications had reported elsewhere. Recently, water-soluble ferulic acid sugar esters from wheat bran had been reported that can stimulate the growth of Bifidobacterium bifidum, and also show excellent antioxidant activity in vitro and in vivo studies. Based on the regard of agricultural wastes re-newly use, conversion of agro-industrial residues to ferulic acid sugar esters not only provides an alternative substrate but also helps to solve some of the problems caused by their accumulation. Coba husk, that abundantly generated from Pu Li, Taiwan, was selected as substrates for ferulic acid sugar esters preparation by acid hydrolysis in this work.
The classical “change-one-factor-at-a-time” approach was first employed to evaluate the effects of hydrolysis conditions, including incubation temperature, TFA concentration and incubation time, on esterified ferulic acid (EFA) producing. Results show the maximum condition for EFA producing was at 100℃, 0.2 M TFA and duration time of 60 min, respectively. A 23 central composition design (CCD) of response surface methodology (RSM), which factors of TFA concentration and incubation time were selected, was further introduced to optimize the condition for EFA preparation. Data from the 10 sets were analyzed to yield regression equations and regression coefficients (R2). From the response surface regression (RSREG) data, the determination coefficient R2 = 0.95 and all variables significantly contributed to this effect. The maximum EFA production predicted by this model is 5.583mg/g from canonical analysis of response surface under the conditions of 0.234 M TFA, at 100℃ and incubated for 59.9 min. After verified the optimum condition, 5.626mg/g EFA were obtained and demonstrated the applicability of this model.
Esterified ferulic acids were prepared based on the above optimal condition. After fractionated by an Amberlite XAD-2 column, fraction obtained by eluting with 50% methanol/water was found to possess 64% EFA recovery. After lyophilized, sample was extracted sequential with methanol and H2O. Approximately 84% EFA of 50% methanol/water elute was found in MeOH extract, and then used for further separation. A preliminary survey of the chromatographic profile of this fraction by reverse phase HPLC analysis using a mobile phase of 20% MeOH/H2O, a major peak with retention time at 24.43 min was observed that comprise 37% of total regression area. In order to isolate this compound, re-chromatographed was conducted by semi-preparative reversed-phase HPLC. A pure compound was isolated as a light yellow powder and identified as feruloyl-L-arabinose (FAA) after intensive NMR and EI mass spectrum analysis and compared to earlier reports.
The antioxidant activity, including total antioxidant activity and toward different reactive oxygen species, of FAA was further investigated. In the TEAC assay, FAA show the 50% inhibition concentration (IC50) value of FAA was 15.86 μg/mL in comparison with the standard antioxidants FA and Trolox with IC50 values at 2.96 and 4.76μg/mL, respectively. The super coil (SC) form in DNA can be converted to the open circular (OC) form due to the hydroxyl radical damage which generated based on the Fenton reaction. In evaluating the protection effect of FAA on oxidation-induced DNA damage study, FAA and FA both exhibited the protective capacity 34.6%~56.6% and/or 58.3~64.4% in the concentration range of 2.5~10μg/mL, respectively. The ORAC assay was further used to test the antioxidative capacity of antioxidants to quench peroxyl radicals. In ORAC assay, FAA had a concentration-dependent increase in the inhibition of fluorescein decay. The antioxidative activity against peroxyl radicals was in the order of FA> FAA > Trolox in this study. This demonstrated FAA could directly scavenge the peroxyl radicals to terminate the free radical chain reaction of lipid peroxidation. Superoxide radicals could promote oxidative reactions and form perhydroxyl radicals which initiate lipid oxidation. FAA could effectively scavenge superoxide radicals (IC50 95.04 ug/mL), however, less than ascorbic acid (IC50 13.67 ug/mL) and FA (IC50 39.02 ug/mL). Based on the above results, FAA and FA can exhibit significantly antioxidant activity especially in peroxyl radical and OH radical scavenging. The antioxidant capacity of FAA slightly less than FA should attribute to its sugar moiety. However, the sugar moiety makes FAA more hydrophilic than FA. Based on this character, FAA should superior than FA that can adapt to industrial application, especially in food industry.
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