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|標題:||Antioxidative properties of lotus seed extract|
single cell gel electrophoresis
|摘要:||本研究探討蓮子不同溶劑(水、正己烷及乙酸乙酯)萃取物之抗氧化性，以及蓮子萃取物對於活性氧與活性氮化物對人體淋巴球細胞及巨噬細胞(RAW264.7) DNA傷害之影響，並探討蓮子萃取物之多酚化合物在抗氧化性所扮演的角色。在抗氧化特性之研究顯示，蓮子水萃取物(0.2 mg/ml)對亞麻油酸之自氧化作用，均具有顯著的抑制效果(IP %均大於96 %)，但對DPPH自由基則無明顯之清除效應。與乙酸乙酯及正己烷萃取物相較，蓮子水萃取物對亞鐵離子之螯合能力(57 %)和過氧化氫的清除能力(44 %)均較佳。
由單細胞電泳試驗結果顯示，蓮子萃取物於選取之濃度範圍內(0.2 ~ 0.6 mg/ml)，對於淋巴球細胞並無毒性效應(細胞存活率皆> 90 %)，亦不會造成淋巴球細胞之DNA損傷，或促使脂質過氧化物生成。此外，對於過氧化氫(H2O2)誘導淋巴球細胞DNA損傷亦具良好的抑制效果，於濃度0.6 mg/ml時，其抑制率為水萃取物(61 %) > 乙酸乙酯萃取物(30 %) > 正己烷萃取物(20 %)。因此蓮子水萃取物能減輕H2O2誘發淋巴球細胞之DNA損傷，可能是其具有螯合金屬離子以及清除過氧化氫等抗氧化特性。
進一步探討蓮子萃取物清除活性氮化物之結果顯示，蓮子萃取物於濃度0.2 mg/ml時具有抑制sodium nitroprusside (SNP)釋放NO之能力，其中以乙酸乙酯及水萃取物的抑制效果較好，而正己烷萃取物的抑制效果較弱。以MTT試驗顯示蓮子萃取物在相同濃度下(0.2 mg/ml)，對RAW264.7均無毒性(細胞存活率皆> 90 %)，而由單細胞電泳試驗結果顯示，蓮子萃取物均不會造成RAW264.7之DNA損傷。乙酸乙酯萃取物、水萃取物及正己烷萃取物於0.2 mg/ml時對於SNP誘發RAW264.7之DNA損傷抑制效果分別為53 %、49 %及35 %。然而與乙酸乙酯及正己烷萃取物相比較，蓮子水萃取物對於LPS (lipopolysaccharide)誘導RAW264.7生成NO及對於tyrosine與 peroxynitrite作用進行nitration形成3-nitrotyrosine均具有較佳之抑制效應。水萃取物、乙酸乙酯萃取物及正己烷萃取物於0.2 mg/ml時對於peroxynitrite誘發RAW264.7之DNA損傷抑制效果分別為63 %、59 %及38 %。
蓮子水萃取物含有最多的總多酚類(143.1 mg/g)，其次為乙酸乙酯萃取物(28.5 mg/g)，正己烷萃取物最低(25.7 mg/g)。以HPLC分析蓮子萃取物之酚酸及類黃酮化合物，結果顯示蓮子水萃取物以酚酸化合物為主，包括p-hydroxybenzoic acid (9.61 mg/g)、gallic acid (9.36 mg/g)、chlorogenic acid (9.72 mg/g)及caffeic acid (9.39 mg/g)；乙酸乙酯萃取物除了gallic acid (2.83 mg/g)及ferulic acid (9.39 mg/g)之外，另含有rutin (6.45 mg/g)之類黃酮化合物；正己烷萃取物中則無酚酸及類黃酮化合物被偵測出。
The objectives of this research were to study the antioxidant activity of lotus seed (Nelumbo nucifera) extracts (LSE), to study the effect of LSE on reactive oxygen species (ROS) and reactive nitrogen species (RNS) induced DNA damage in human blood lymphocytes and macrophage RAW264.7, and to study the polyphenolics in LSE in relation to its antioxidant activity. The results showed that LSE (0.2 mg/ml) inhibited the peroxidation of linoleic acid (IP > 96 %), whereas they showed no scavenging effect on DPPH. The water extract of lotus seed (LSWE) showed a better chelating effect on Fe2+ (57 %) and scavenging effect on H2O2 (44 %) as compared with ethyl acetate extract (LSEAE) and n-hexane extract (LSHE) of lotus seed. The effect of LSE on DNA damage in human lymphocytes was investigated by means of single cell gel electrophoresis (SCGE). LSE alone showed no cytotoxicity (cell viability > 90 %) and did not cause DNA damage or the production of lipid hydroperoxide toward human lymphocytes under a concentration of 0.2-0.6 mg/ml. However, LSE reduced the H2O2-induced DNA damage in human lymphocytes in a dose-dependent manner. At a concentration of 0.6 mg/ml, the inhibitory effect of LSE on H2O2-induced DNA damage in lymphocytes was in the order LSWE (61 %) > LSEAE (30 %) > LSHE (20 %). The inhibitory effect of LSWE on H2O2-induced DNA damage in lymphocytes might be due to the chelating ability of Fe2+ and to the savenging effect of H2O2. The reactive nitrogen species (RNS) scavenging capacity of the LSE was also evaluated. The LSE decreased nitric oxide (NO) production from sodium nitroprusside (SNP) in a concentration dependent manner. The inhibitory ability of the extracts was in the order LSEAE > LSWE > LSHE. LSE (0.2 mg/ml) showed no cytotoxicity to macrophage RAW264.7 by MTT assay (cell viability > 90 %). Incubation of LSE alone also did not cause DNA damage in RAW264.7. The protective effect of LSEAE, LSWE, and LSHE at a concentration of 0.2 mg/ml towards DNA damage in RAW264.7 induced by SNP was 55 , 49, and 35 % , respectively. Compared with LSEAE and LSHE, LSWE had a greater potential protect effect against peroxynitrite-dependent nitration of tyrosine to 3-nitrotyrosine and inhibited LPS-stimulated NO generation on RAW264.7. The inhibitory effect of LSWE, LSEAE and LSHE at a concentration of 0.2 mg/ml towards peroxynitrite-induced DNA damage in RAW264.7 was 63 , 59, and 38 %, respectively. LSWE contains the highest polyphenol content (143.1 mg/g), followed by LSEAE (28.5 mg/g) and LSHE (25.7 mg/g). The phenolic acids and flavonoids in LSE were further determined by means of HPLC. It was found that phenolic acid was the main compound in LSWE, including p-hydroxybenzoic acid (9.61 mg/g), gallic acid (9.36 mg/g), chlorogenic acid (9.72 mg/g) and caffeic acid (9.39 mg/g). LSEAE contains gallic acid (2.83 mg/g), ferulic acid (9.39 mg/g), and a flavonoid rutin (6.45 mg/g). No phenolic acid and flavonoids were detected in LSHE. In conclusion, LSE had antioxidant activity, and LSWE exhibited the most antioxidant activity. Since LSWE contains the greatest amount of polyphenol, polyphenol may play an important role to the antioxidant activity of LSWE.
|Appears in Collections:||食品暨應用生物科技學系|
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