抗氧化活性評估之新巨噬細胞模式

Novel Macrophage Cell Model for Antioxidative Activity Evaluation

許榮廷(Teeradate Kongpichitchoke)

指導教授 ： 黃卓治 ； 邱亞伯 ； 徐睿良

國立屏東科技大學/國際學院/熱帶農業暨國際合作系/博士(2015年)

https://doi.org/10.6346/NPUST.2015.00087

摘要

氧化壓力曾被證實引起許多慢性疾病。在免疫系統中，過量超氧化合物為氧化壓力主要來源。現今，許多科學家正尋找天然物中含有高效能氧化物之有效成分。在酚類化合物中，黃酮類化合物(flavonoids)曾被報導具有高抗氧化活性。然而，黃酮類化合物之結構及其抗氧化活性關係仍舊不清楚。因此，本研究目PMA活化小鼠巨噬細胞(RAW264.7)藉由體外實驗(in-vitro)及電腦軟體模擬(in-silico)檢測分析發展新穎細胞模式之抗氧化活性評估。三種黃酮類化合物之B環上含不同數量之羥取代基(hydroxyl groups)，包括生松素(pinocembrin)、柚皮素(naringenin)及聖草酚(eriodictyol)，被用於初始化合物樣品之創建模式。三種化合物受到DPPH自由基清除活性，體外實驗評估其活性含氧物(ROS)及蛋白免疫印跡分析；而分子嵌合研究以電腦軟體模擬分析。體外實驗研究結果得知，聖草酚抗氧化活性較生松素及柚皮素明顯佳(p ＜ 0.05)。生松素、柚皮素及聖草酚之活性含氧物抑制，其IC50分別為44.9 ± 0.57、30.2 ± 0.61及17.4 ± 0.40 μM。而蛋白免疫印跡分析之結果顯示三種黃酮類化合物(10 μM)可抑制PKCδ與p47phox磷酸化，其聖草酚抑制活性較生松素及柚皮素佳。而分子嵌合試驗結果得知聖草酚B環上之兩個羥取代基與結合PKCδ佛波酯結合位中扮演重要角色。而聖草酚、柚皮素及生松素之-CDOCKER-能量值分別為21.49、16.95與15.22。因此，黃酮類化合物藉由兩種方式測定抗氧化活性，分別為直接清除超氧化合物及佔據PKCδ佛波酯結合位。

兩種生物活性化合物，未具黃酮類結構，來自加工天然產品，為乾燥四季桔(Citrofortunella microcarpa)之果皮與種子，及臺灣茶，測定其細胞模式之抗氧化活性。本研究藉由HPLC、LC-MSn及NMR實驗測定乾燥與發酵後之四季桔和臺灣茶其生物活性化合物之含量。結果顯示，四季桔及臺灣茶之5-羥甲基糠醛(HMF)與沒食子酸(gallic acid)於食物加工過程中具強烈影響。新鮮四季桔缺乏HMF，然而放置於70℃烘箱4天，乾燥後成熟之四季桔果皮及種子顯示最高含量(0.86 mg/g乾重)。臺灣茶具不同發酵程度，分別為非發酵(綠茶)、部分發酵(烏龍茶)、全發酵(紅茶)及後發酵(普洱茶)，測定其沒食子酸含量。而綠茶、烏龍茶、紅茶及普洱茶之沒食子酸含量分別為1.67 ± 0.06、1.92 ± 0.56、6.09 ± 0.06與22.0 ± 1.03 mg/g。再者，10 μM HMF及沒食子酸加於細胞模式中並評估其抗氧化活性。而HMF及沒食子酸之自由基清除活性，分別為37.8 ± 6.40 與19.3 ± 3.92%。然而，HMF及沒食子酸可明顯抑制活性含氧物和PKCδ與p47phox磷酸化；而HMF及沒食子酸之抑制活性卻無明顯差異。最後，HMF及沒食子酸之-CDOCKER-能量值分別為18.23與15.94，其顯示兩種化合物可預防佛波酯活化PKCδ，因此其具有抗
氧化活性。

關鍵字

抗氧化活性 ； 生物活性化合物 ； DPPH ； 活性氧 ； PKC ； p47phox ； 分子嵌合

並列摘要

Oxidative stress has been proven to cause many chronic dieseases. Overproduction of superoxide compounds in immunological system is well-known as a source of oxidative stress. Nowadays, many scientists are looking for effective compounds containing high antioxidant potency from natural products. Among them, phenolic compounds, especially flavonoids, have been reported for their high antioxidative activity. However, the relationship between the flavonoid structure and its antioxidative activity remains unclear. The main objective of this study was to report a novel cell model for antioxidative activity evaluation by using both in-vitro and in-silico assays with PMA-activated murine macrophage cell (RAW264.7). First, three flavonoids, containing different number of hydroxyl groups on B-ring of flavonoid including pinocembrin (0 group), naringenin (1 group), and eriodictyol (2 groups), were used as initial compound samples for creating the model. All three compounds were subjected to DPPH scavenging activity, reactive oxygen species (ROS) and proteins immunoblotting assays for the in-vitro assays, and in-silico assay by molecular docking study. From all in-vitro experiments, eriodictyol had significant higher antioxidative activity over pinocembrin and naringenin (p < 0.05). The IC50 values for ROS inhibition by pinocembrin, naringenin and eriodictyol were 44.9 ± 0.57, 30.2 ± 0.61, and 17.4 ± 0.40 μM, respectively. Moreover, proteins immunoblotting assays showed all three flavonoids in this study at 10 µM concentration could inhibit phosphorylation of PKCδ and p47phox in which eriodictyol performed remarkable inhibition activity over pinocembrin and naringenin. Lastly, molecular docking showed that two hydroxyl groups on the B-ring of eriodictyol played crucial role in binding with PKCδ phorbol ester binding site. The –CDOCKER_energy values of eriodictyol, naringenin, and pinocembrin were 21.49, 16.95, and 15.22, respectively. Therefore, flavonoids exhibited their antioxidative activity by two methods; directly scavenging superoxide compounds, and occupying the phorbol ester binding site of PKCδ.

The antioxidative activity of two bioactive compounds, without flavonoid-like structure, from two processed natural products; dried calamondin (Citrofortunella microcarpa) peels and seeds, and Taiwan teas, was determined using the cell model. Firstly, effect of drying and fermentation process on calamondin and Taiwan teas, respectively, on bioactive compound content were investigated by using HPLC, LC-MSn and NMR. It was found that 5-hydroxymethyl furfural (HMF) and gallic acid in calamondin, and teas, respectively, were greatly affected by the food processes. HMF was originally absent in fresh calamondin, but the highest amount (0.86 mg/g dry weight) was for the first time found in dried mature calamondin peels and seeds from drying at 70 ºC for four days. Taiwanese teas with different fermentation degree of tea; non-fermented (green tea), semi-fermented (oolong tea), full-fermented (black tea), and post-fermented (pu-erh tea) were determined for gallic acid content. The amount of gallic acid found in green, oolong, black and pu-erh tea were 1.67 ± 0.06, 1.92 ± 0.56, 6.09 ± 0.06, and 22.0 ± 1.03 mg/g, respectively. Secondly, both HMF and gallic acid at 10 µM concentration were subjected to the cell model for antioxidative activity evaluation. Radical scavenging activity of HMF and gallic acid were 37.8 ± 6.40 and 19.3 ± 3.92%, respectively. Although both HMF and gallic acid could significantly inhibit the ROS production, and phosphorylation of PKCδ and p47phox (p < 0.05), insignificantly different activity between HMF and gallic acid were reported (p > 0.05). Lastly, the –CDOCKER_energy of HMF and gallic acid were 18.23 and 15.94, respectively, reflecting that both compounds exhibited their antioxidative activity by preventing PKCδ to be activated by phorbol esters.

並列關鍵字

Antioxidative activity ； bioactive compound ； DPPH ； reactive oxygen species ； PKC ； p47phox ； molecular docking

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