探討海參水萃物對小鼠黑色素瘤細胞株和斑馬魚胚胎之抗黑色素生成效果

海參乾貨製程中水萃物過去常作為廢棄物丟棄，有研究發現其中成分比例和活體海參相仿。本實驗分別使用台灣水域與日本水域之仿刺參在加工後產生的水萃物(sea cucumber aqueous extract；SCAE)經凍結乾燥處理後回溶，再對小鼠黑色素瘤細胞株(B16F10 cell) 與斑馬魚胚胎進行實驗，觀察抑制黑色素生成的效果，記錄半致死劑量、黑色素生成量、黑色素生成相關蛋白質與基因表現量，最後進行定性與定量。實驗結果顯示在凍結乾燥後的SCAE-TW或SCAE-JP對細胞毒性實驗發現100μg/mL以上濃度均對細胞與斑馬魚胚胎產生傷害使活存率下降，而10μg/mL以下濃度細胞與斑馬魚胚胎活存率和控制組相近且沒有顯著差異，計算其半致死濃度(LC50)分別為54μg/mL與201μg/mL。細胞實驗結果顯示，隨著SCAE-TW與SCAE-JP使用濃度越高，實驗組黑色素生成量與正控制組相比顯著減少。酪胺酸脢活性同樣隨著SCAE-TW與SCAE-JP使用濃度越高，與正控制組別相比也顯著減少。細胞之黑色素生成相關基因MITF和TYR在SCAE-TW與SCAE-JP在 10μg/mL、1μg/mL、0.1μg/mL三種劑量下表現量與正控制組相比均有顯著下降；而TRP1與TRP2基因與正控制組相比雖然有下降趨勢，卻沒有顯著統計差異。斑馬魚實驗結果顯示黑色素生成量在SCAE-TW與SCAE-JP 於10μg/mL濃度下與控制組相比有顯著下降，酪胺酸脢活性實驗結果類似於細胞實驗，顯示隨著使用的SCAE濃度越高，產生量顯著減少。SCAE-TW或SCAE-JP皆具有易起泡且泡沫不易消退的特性，由foam test的泡沫穩定性與saponin定量實驗結果得知，推測日本海域的仿刺參的水煮液之saponin成分含量皆高於台灣海域仿刺參水煮液，並在與皂皮樹saponin標準品建立之標準曲線進行外插法計算後，推算SCAE-TW與SCAE-JP每100mg分別含有3.45mg和6.48mg的saponin化合物。SCAE-TW與SCAE-JP經過本篇研究證實SCAE可能含有皂素化合物成分，藉由抑制黑色素生成途徑之相關基因表現與蛋白質活性，達到抑制黑色素生成之效果。

關鍵字

仿刺參；水萃液；黑色素生成；小鼠黑色素瘤細胞株；斑馬魚胚胎

並列摘要

The sea cucumber is an economically important fishery species and have long been used as food and traditional medicine. In the processing of sea cucumber dry product, the aqueous extract byproducts are discarded as waste, but some studies had indicated that the composition of aqueous extract is similar to live sea cucumber itself. In the present study, we first conduct experiments on the B16F10 cell line and zebrafish embryos, then investigated the property of sea cucumber (Apostichopus japonicus) aqueous extract (SCAE), obtained from Taiwan and Japan aquafood factory. The survival rate experiments indicate that SCAE-TW or SCAE-JP concentration after lyophilization treatment at 100μg/mL or above is harmful to the B16F10 cells and zebrafish embryos, and their lethal concentration 50% are 54μg/mL and 201μg/mL, respectively；on the contrary, concentration at 10μg/mL or lower then show no significant difference to control group. The cell and zebrafish experiments reveal that as concentration of SCAE-TW or SCAE-JP increases, the melanin content and tyrosinase activity decreased in a dose dependent manner. Melanogenesis related genes such as MITF and TYR gene expression in the B16F10 cells were significantly inhibited by the treatment of SCAE-TW or SCAE-JP by the quantitative PCR analysis. The foam test reveals that SCAE from either Taiwan or Japan are possessed with the feature of amphiphilic molecule similar to saponin compound. According to the standard curve of Quillaja bark saponin standard, we calculated SCAE-TW and SCAE-JP contains 3.45mg and 6.48mg of the saponin component per 100mg after lyophilization treatment, respectively. This study demonstrated that the saponin compounds in SCAE may achieve anti-melanogenesis through the inhibition of melanogenesis related gene expression and protein activity.

並列關鍵字

sea cucumber (apostichopus japonicus ； aqueous extract ； melanogenesis ； murine B16F10 cell line ； zebrafish embryos

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