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Studies on Antioxidant functionality of Hsian-tsao (Mesona procumbens Hemsl.)
|關鍵字:||仙草;Hsian-tsao (Mesona procumbens Hemsl.);抗氧化性;活性氧與自由基;人類肝細胞株;氧化傷害;脂質過氧化;分離純化;酚類化合物;antioxidative activity;reactive oxygen species and free radical;Chang liver cells;oxidative damage;lipid peroxidation;isolation and identification;phenolic component||出版社:||食品科學系||摘要:||
中 文 摘 要
本研究主要是探討仙草 (Mesona procumbens Hemsl.)的抗氧化機能性以及其應用。第二章探討仙草水萃取物之抗氧化特性，結果顯示仙草水萃取物在亞麻油酸系統中具有良好抗氧化性，其抗氧化性與總多酚類含量之間呈現正相關性 (p < 0.05)。仙草水萃取物在100-200 mg/mL濃度下，清除DPPH自由基、超氧陰離子、過氧化氫、一氧化氮及過氧化自由基等方面都具有顯著的效果，其抑制率為50-90 % 之間，且呈劑量-反應關係。另外，仙草水萃取物具有抑制由鐵離子所誘導紅血球細胞膜傷害，以及過氧化氫所誘導肝細胞脂質過氧化的能力，在200 mg / mL濃度下，其氧化抑制率分別為55.7及46 %。仙草水萃取物在人類肝細胞株中並未顯現出細胞毒性及基因毒性，於100 mg/mL 濃度下也具有抑制由過氧化氫所誘導肝細胞DNA氧化傷害的效果，其氧化抑制率可達88.5 %。仙草水萃取物於100 mg/mL濃度下，具有77.6 %清除胞內活性氧的能力，且呈濃度-劑量反應。由上述結果顯示，仙草水萃取物具有保護肝細胞，避免氧化傷害的效果。
第三章則針對仙草之抗氧化成分加以分離純化。結果顯示仙草 (Mesona procumbens Hemsl.) 之不同溶劑萃取物以乙酸乙酯萃取物抑制亞麻油酸氧化的效果最佳，抑制率高達93 %。將乙酸乙酯萃取物進行分離純化，並以EI-MS，1H 及 13C-NMR光譜分析加以鑑定，獲得類黃酮化合物kaempferol、apigenin，以及固醇衍生物stigmasterol、b-sitosterol、oleanolic acid 及 ursolic acid 等六種純化物質。此六種純化物質與BHA 及a-生育醇在200 mg/mL 濃度下，對亞麻油酸的氧化抑制率依序為BHA = kaempferol = 仙草乙酸乙酯萃取物 > a-生育醇 > ursolic acid > oleanolic acid > apigenin > b-sitosterol > stigmasterol。Kaempferol 在純化物質中顯現最佳之抗氧化性及最高的含量，顯示kaempferol為仙草乙酸乙酯層之主要抗氧化成分。
第四章主要探討仙草酚酸化合物的分離純化與鑑定。仙草75 %甲醇萃取物在不同溶劑萃取區分中，以酸性乙酸乙酯萃取物 (pH 2) 具有最高的總多酚類含量以及極佳的抗氧化性。經由分離純化，以及UV、EI-MS、1H-NMR及13C-NMR等光譜鑑定後，得知五個純化物質均屬酚酸類。此五種純化物質與BHA 及a-生育醇在200 mg/mL 濃度下，對亞麻油酸的氧化抑制率依序為BHA = Trolox > 酸性乙酸乙酯萃取物 > caffeic acid > a-tocopherol > protocatechuic acid > syringic acid > vanillic acid > p-hydroxybenzoic acid.，顯示酚酸類在仙草之抗氧化性中扮演重要角色。其中以caffeic acid顯現最佳之抗氧化性以及最高含量，為仙草75 %甲醇萃取物之主要抗氧化成分。
第五章探討仙草經過加鹼水煮處理對其抗氧化性及酚酸化合物之影響。仙草添加不同濃度 (0.1-1.5 %)之鹼液 (碳酸鈉、碳酸氫鈉)，加熱0.5-3 小時後進行分析。結果得知以添加0.1 -0.3 %碳酸鈉鹼液，加熱2小時下所得萃取物具有較高的多酚類含量與抗氧化效果，並發現仙草鹼水煮萃取物之抗氧化性、清除自由基的能力與多酚類含量有高度正相關性。仙草鹼水煮萃取物抗氧化能力會受到過量鹼液以及長時間加熱處理而破壞。在酚酸化合物含量的變化方面，發現隨鹼液濃度提高，各酚酸類化合物含量有下降趨勢，其中尤以含量最高的caffeic acid變化也最為明顯。添加碳酸鈉鹼液處理對仙草抗氧化性有較佳保留效果。另外，亦探討在不添加鹼液的不同加工方式處理下，對其清除自由基的能力與多酚類含量的影響。仙草經浸泡18小時後再以121℃，20分鐘之殺菌釜處理，為清除自由基的最適當加工條件。殺菌釜處理下所得到的酚酸化合物萃取率，特別是caffeic acid含量，高於以水煮加熱的處理方式，顯示殺菌釜處理優於水煮加熱的處理條件。
第六章探討kaempferol等七種分離自仙草之酚類化合物的抗氧化特性。Kaempferol (200 mg/mL)抑制亞麻油酸氧化的效果最佳，與同濃度BHA、Trolox及仙草水萃取物之抗氧化性相近，caffeic acid 次之，並優於生育醇。Caffeic acid及kaempferol 在200 mg/mL濃度下清除 DPPH自由基能力也達96 及93 %，顯示此兩種化合物為提供仙草抗氧化性的重要成分。Caffeic acid、kaempferol及protocatechuic acid具有抑制過氧化氫誘導紅血球細胞膜以及肝細胞脂質過氧化的效果。酚類化合物在人類肝細胞株中並未顯現出細胞毒性及基因毒性，並具有降低由過氧化氫誘導DNA氧化傷害的效果。Kaempferol 及caffeic acid 在10 mM濃度下便分別有清除人類肝細胞株胞內活性氧31.2 與51.7 %的效果。
This study evaluated the antioxidant functionality of Hsian-tsao (Mesona procumbens Hemsl.). Chapter 2 focuses on the investigation of the antioxidative activities of the water extracts of Hsian-tsao. The results showed that the water extracts of Hsian-tsao have antioxidative activities in linoleic acid peroxidation system and correlated with the polyphenol contents (p < 0.05). The water extracts of Hsian-tsao have the positive concentration-dependent scavenging effect on DPPH radical, superoxide anion, hydrogen peroxide, nitric oxide and peroxyl radical. Furthermore, the water extracts of Hsian-tsao have the inhibitory effect on oxidative damage to biomolecules. The extracts also exhibited 55.7 and 46 % inhibitory effect, respectivity, on the lipid peroxidation of ghost memberane and Chang liver cells induced by hydrogen peroxide, under the concentration of 200 mg/mL. No toxicity was found in the water extracts of Hsian-tsao towards Chang liver cells, and the cell viability was over 90 %. The water extracts of Hsian-tsao could inhibit 88.5 % oxidative DNA damage induced by hydrogen peroxide in Chang liver cells under the concentration of 100 mg/mL. The water extracts of Hsian-tsao reduced 77.6 % of intracellular reactive oxygen species (ROS) in Chang liver cells at a concentration of 100 mg/mL. Thus, the water extracts of Hsian-tsao have protective effect against oxidative damage in Chang liver cells.
Chapter 3 focused on the extraction and identification of antioxidant components from Hsian-tsao. The results showed that the antioxidant activities of ethyl acetate extracts of Hsian-tsao, which exhibited 93 % inhibition on the peroxidation of linoleic acid, was greater than those of BHA and a-tocopherol at a same concentration of 200 mg/mL. The ethyl acetate extracts of Hsian-tsao was further separated and isolated by the preparative HPLC equipped with a silica column. Six components were identified by UV, EI-MS, 1H-, and 13C-NMR. These compounds included flavonoids, kaempferol and apigenin, and sterol derivatives (stigmasterol, b-sitosterol, oleanolic acid, and ursolic acid). The antioxidant activity for those compounds was in the order of BHA = kaempferol = ethyl acetate extract > a-tocopherol > ursolic acid > oleanolic acid > apigenin > b-sitosterol > stigmasterol. Kaempferol was the major compound with the strongest antioxidant activity and the highest content in the ethyl acetate extracts of Hsian-tsao.
Chapter 4 studied the purification and identification of the phenolic acids in the extracts of Hsian-tsao. The Hsian-tsao was extracted with 75 % methanol, and then the extracts were fractionated with different polarities of solvents. The results showed that the fraction obtained with acidic ethyl acetate (pH 2) possessed the highest content of the phenolic compounds and the strongest antioxidant activity on the peroxidation of linoleic acid. The fraction obtained with the acidic ethyl acetate was separated and identified as protocatechuic acid , p-hydroxybenzoic acid, vanillic acid, caffeic acid, syringic acid by UV, EI-MS, 1H and 13C-NMR. The antioxidant activity of those compounds was in the order of BHA = Trolox > acidic ethyl acetate extract of Hsian-tsao > caffeic acid > a-tocopherol > protocatechuic acid > syringic acid > vanillic acid > p-hydroxybenzoic acid. These results suggested that the phenolic acids act as the most important antioxidant components in 75 % methanol extract of Hsian-tsao, especially the caffeic acid.
Chapter 5 aimed to investigate the effects of processing conditions on the antioxidative activity and the changes in phenolic acids of the extracts from Hsian-tsao. Hsian-tsao was extracted with boiling water for 0.5-3 h by the addition of 0 to 1.5 % of Na2CO3 or NaHCO3 solution. A high content of total phenolic acid and stronger antioxidative activity were found in the extracts from the Hsian-tsao treated with Na2CO3 at concentrations of 0.1-0.3 % under heating for 2 h. The antioxidative activity, the scavenging activity on DPPH radical and superoxide anion, and the content of total phenolic compounds of Hsian-tsao extracts decreased with the increases in the alkaline concentration and the heating time. In addition, the content of the phenolic acid, especially the caffeic acid, was significantly decreased with the increase of the alkaline concentration. The effect of NaHCO3 on decreasing the content of phenolic acid was greater than that of Na2CO3. The antioxidative activity and the scavenging effects on DPPH radical and superoxide anion of Hsian-tsao extracts were highly correlated with the total phenolic contents of the extracts. In addition, the effect of the processing conditions without alkaline treatment on the free radical scavenging effects and phenolic acid contents of the extracts from Hsian-tsao was also evaluated. The Hsian-tsao soaked at room temperature for 18 h and then autoclaved at 121℃ for 20 min to obtain the extracts. The contents of phenolic acid and caffeic acid in Hsian-tsao water extracts obtained by autoclave sterilization retort was higher than that by boiling water.
Chapter 6 focused on the antioxidant properties of seven phenolic compounds in the extracts of Hsian-tsao. Kaempferol showed the strongest antioxidant activity on the peroxidation of linoleic acid, which was equal to those of BHA, Trolox and the water extract of Hsian-tsao under the same concentration of 200 mg/mL. Caffeic acid was the second one and greater than a-tocopherol. The scavenging effect of kaempferol and caffeic acid on DPPH radical were 93 and 96 %, respectivity, at a concentration of 200 mg/mL. Caffeic acid, kaempferol and protocatechuic acid had marked inhibitory effect on lipid peroxidations in the ghost membranes and Chang liver cells induced by hydrogen peroxide. No toxicity was found in the Chang liver cells treated with the phenolic compounds of Hsian-tsao. The cell viability was over 90 %. The phenolic compounds of Hsian-tsao could inhibit oxidative DNA damage in Chang liver cells induced by hydrogen peroxide. The intracellular reactive oxygen species (ROS) of Chang liver cells was decreased for 31.2 and 51.7 % by caffeic acid and kaempferol, respectivity, at a concentration of 10 mM. Thus, the caffeic acid and kaempferol, the highest content of phenolic compounds in the extracts of Hsian-tsao, were the major antioxidant compounds for the scavenging effects on free radical, inhibitory biomembrane and oxidative DNA damage, and the decreasing of intracellular reactive oxygen species in Chang liver cells.
Keyword: Hsian-tsao (Mesona procumbens Hemsl.), antioxidative activity, reactive oxygen species and free radical, Chang liver cells, oxidative damage, lipid peroxidation, isolation and identification, phenolic compound.
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