Approach to Antioxidant Activity and Related Compounds in Hot Water Extract of Freshwater Clam

• Main Authors: Hsin-Yu Chen, 陳星宇
• Other Authors: Shyi-Neng Lou
• Format: Others
• Language: zh-TW
• Published: 2008
• Online Access: http://ndltd.ncl.edu.tw/handle/13012926739634787720

碩士 === 國立宜蘭大學 === 食品科學系碩士班 === 97 === Hot water extract of freshwater clam (Corbicular fluminea) was traditionally claimed hepatoprotective activity, antioxidant activity, anti-cancer, anti-inflammatory effect and other relevant physiological functions. The aim of this study was to evaluate the effect of extraction on antioxidant activity and to separate and collect the extract by semi-preparative chromatography. The obtained fractions were subjected to the HPLC analysis to identify the major antioxidant compounds. The results indicated that DPPH radical scavenging effect, reducing power, and chelating Fe2+ ability of hot water extracts increased with increasing concentration. The lowest values of 50% inhibitory concentration (IC50) on DPPH radical scavenging effect (1.82 mg/mL) and reducing power (10.53 mg/mL) were found in 40 min extract. The highest reducing effect of IC50 on DPPH radical scavenging effect and reducing power were observed during 20 to 40 min cooking. The decreasing ratio were 0.023 and 0.733 IC50/mg/mL, respectively. Therefore, the extract of 40 min cooking was selected and subjected to the following analysis. The extracts of Cultured freshwater clam (CFC) and Field freshwater clam (FFC) after cooking for 40 min were separated to four fractions by a superpose 12 prep. gel filtration chromatography. The fraction molecular weight range was Fraction A ( > 1300 Da), Fraction B (1300-630 Da), Fraction C (630- 240 Da) and Fraction D (< 240 Da). The DPPH radical scavenging potency of fraction B and C were 20.28 and 18.99 %/mg/mL in CFC, while these were 21.03 and 17.22 %/mg/mL in FFC, respectively. The fraction C was chosen to identify the antioxidant compounds by semi-preparative chromatography method. Nine fractions of the obtained CFC fraction C (CFC-HW40-C) were separated by semi-preparative reversed-phase HPLC. The fractions of CFC-HW40-C-IV、III and VIII had high DPPH radical scavenging potency with the value of 323.40, 140.134, and 145.98 %/mg/mL, respectively. The sum of these three fractions contributed 60 % antioxidant activity in the fraction of CFC-HW40-C. Similarly, five fractions of FFC-HW40-C were collected. The DPPH scavenging potency of three fractions, such as FFC-HW40-C-V, III and II, contained high value, which equal to 83.94, 71.44, and 64.61 %/mg/mL, respectively. The 80% antioxidant activity of FFC-HW40-C was due to these three fractions. It was found that the 5`-UMP and 5`-IMP existed in fractions of CFC-HW40-C-III and IV, respectively. The fraction of CFC-HW40-C-VI consisted two nucleotides, such as 5`-AMP and 5`-GMP. However, the main peak in fraction of CFC-HW40-C-IX was identified as tyrosine. For the fraction of FCF-HW40-C, 5`-UMP and 5`-IMP were found in the fraction of FFC-HW40-C-II, 5`-AMP and 5`-GMP in the fraction of FFC-HW40-C-III. Tyrosine was the major peak in the fraction FFC-HW40-C-V. After compare with the standard compounds for the DPPH radical scavenging effect, tyrosine indeed contained DPPH radical scavenging ability, while no DPPH radical scavenging ability of nucleotodes were observed. This implied that the tyrosine related compounds might play an important role for the antioxidant activity in freshwater clam.