|Abstract: ||本研究探討以酵素水解所得耳突麒麟菜 (Eucheuma cottonii) 熱萃多醣液之寡醣區分物，在抗氧化、抗老化、美白、及保濕等方面之生理功效。耳突麒麟菜以 121oC 和 40 min 熱萃所得多醣液，經 5 unit/mL Cellulase (37oC, pH 5.0) 及 5 unit/mL Crude agarases (40oC, pH 6.2 與 6.0) 二組酵素反應所得寡醣水解液之還原醣量最高，再以超過濾膜劃分出八組不同分子質量耳突麒麟菜寡醣 (Eucheuma cottonii oligosaccharides, EUOS) 區分物，測定抗氧化活性。結果顯示，DPPH自由基清除能力及亞鐵離子螯合能力以 3-5 kDa Crude agarases-EUOS 之測試結果 53.15 ± 0.13% (相當於 96.09 ± 0.23 ppm Ascorbic acid) 及 85.22 ± 0.23% (相當於 165.71 ± 0.44 ppm Ascorbic acid) 最好；還原能力測定，寡醣水解物之分子質量越大，還原力越高；總酚含量測定，以 > 5 kDa Crude agarases-EUOS 最高，相當於 104.74 ± 3.21 ppm Gallic acid；其次為 3-5 kDa Crude agarases-EUOS，相當於 84.37 ± 1.28 ppm Gallic acid。選定抗氧化活性較好之 3-5 kDa 及 > 5 kDa Crude agarases-EUOS，進行抗老化、美白功能及保濕實驗。抗老化細胞實驗中，分子質量 3-5 kDa EUOS 對於老鼠胚胎纖維母細胞 NIH 3T3 增生較好；培養 48 hr 對於 NIH 3T3 細胞存活率有增加的作用。3-5 kDa 及 > 5 kDa EUOS，在濃度 125 μg/mL 以上，細胞存活率較高，對於人類皮膚纖維母細胞 CCD-966SK 有增生趨勢。美白功能試驗顯示，對於體外酪胺酸酶活性抑制率以 > 5 kDa EUOS 較好，當濃度 20 mg/mL 時抑制率達 84.83 ± 3.33%，在細胞實驗中 > 5 kDa 及 3-5 kDa EUOS 培養 24 hr 對黑色素腫瘤細胞 A375 無增生作用，培養 48 hr 後，以 > 5 kDa EUOS 在低濃度抑制細胞生長效果較 3-5 kDa 好。皮膚保濕試驗結果顯示，塗抹 > 5 kDa EUOS 後，可增加正常皮膚含水率為約 4.17%；在以 20% SDS 誘發之乾性皮膚角質層含水率， > 5 kDa EUOS可增加約 4.34%，接近甘油效果 (增加 4.83%)。皮膚平滑度測定︰正常皮膚與誘發乾性皮膚之皮膚平滑度測定結果皆是以塗抹 > 5 kDa EUOS 後效果最好，但試驗組間的數據並未呈現明顯差異性。綜合上述耳突麒麟菜寡醣混合物具開發做為抗氧化、美白及保濕功能之化粧品原料深具潛力。|
This study is to explore the efficacy of antioxidative, anti-aging, whitening, and moisturizing of the oligosaccharides produced from Eucheuma cottonii polysaccharides extract digested by enzymes. Polysaccharides of Eucheuma cottonii was extracted by hot water at 121oC for 40 min and then digested by 5 unit/mL cellulase (37°C, pH 5.0) or 5 unit/mL crude agarases (40°C, pH 6.2 and 6.0) obtaining two groups of Eucheuma cottonii oligosaccharide (EUOS) of the highest reducing suger content. These EUOS were then subject to ultrafiltration for separation by molecular weight cut-off membrane into eight different groups for determining antioxidant activity. The best results showed the scavenging effect on DPPH free radical and the ability of chelating on Fe2+ ion for 3-5 kDa crude agarases-EUOS were 53.15 ± 0.13% and 85.22 ± 0.23%, which are equivalent to 96.09 ± 0.23 ppm ascorbic acid and 165.71 ± 0.44 ppm ascorbic acid, respectively. In terms of reducing power test, it was found that the greater the molecular weight of EUOS, the higher the reducing power. Total phenolic content for > 5 kDa crude agarases-EUOS is equivalent to the performance of 104.74 ± 3.21 ppm gallic acid was the highest in the tested EUOS. Followed by the 3-5 kDa crude agarases-EUOS, which showed that it’s total phenolic content equal to 84.37 ± 1.28 ppm gallic acid. Based on the antioxidant activity performaned 3-5 kDa and > 5 kDa crude agarases-EUOS were selected for anti-aging, whitening, and moisturizing capacities experiments. In the anti-aging cell experiments, 3-5 kDa EUOS used for mouse embryonic fibroblast NIH 3T3 cell proliferation performed better than > 5 kDa EUOS. In addition, 48 hr culturing of NIH 3T3 cells with 3-5 kDa EUOS increased cell survival counts. EUOS 3-5 kDa and > 5 kDa fractions which used in the concentrations of 125 μg/mL or more, led to a higher survival rate for human skin fibroblast CCD-966SK terd to be hyperplasia. Whitening function tests showed that the in vitro tyrosinase inhibition rate of > 5 kDa EUOS was better than that of 3-5 kDa EUOS. When the concentration of EUOS was at 20 mg/mL, the inhibition rate for tyrosinase activity was 84.83 ± 3.33%. For melanoma cell growth inhibition experiments, EUOS > 5 kDa and 3-5 kDa fractions were applied to cultured melanoma cells A375. Over a 24 hr incubation period, there was no proliferation of cells in the culture. Which incubated to 48 hr, it was observed that > 5 kDa EUOS fraction at low concentrations was more effective in inhibiting melanoma cell growth than 3-5 kDa EUOS did. With regards to moisturizing capacity, > 5 kDa EUOS present a high water content of normal skin than control by 4.17%, which was better than 3-5 kDa EUOS had. In 20% SDS-induced dry skin stratum corneum, water content of the > 5 kDa EUOS group was increased to about 4.34%, close to the effect of glycerin (an increase of 4.83%). For skin smoothness, > 5 kDa EUOS showed the best results in terms of improving dry skin and normal skin. However, the results of these experiments showed no obvious improvement over those for the control group. It is evident from this study that derived oligosaccharides from Eucheuma cottonii are useful in antioxidative, whitening, and moisturizing shin and show good potential to be raw material for cosmetics.