| 摘要: | 腸泌素是由腸道所產生的胜肽荷爾蒙，可以加強葡萄糖依賴性胰島素的分泌，除具有抑制昇糖激素、減慢胃排空及抑制食慾等降血糖作用外，更可以避免傳統口服降血糖藥物為病人帶來低血糖及體重增加等副作用。但incretin在身體的半衰期約2分鐘，會被雙胜肽水解酶 (Dipeptidyl peptidase-IV；DPP-IV) 酵素所水解，失去原來的活性。目前有研究指出藉由抑制內生性 DPP-IV活性是治療第2型糖尿病的新方向。
本實驗探討從鮪魚蒸煮液蛋白質水解物對抑制DPP-IV活性之能力，從中篩選出可以抑制DPP-IV之活性胜肽及鑑定其胺基酸序列。使用2種不同濃度 (1和2 mg/mL) 商業酵素 [ Protease XXIII (PR)、Orientase 90N (OR) ] 在最適條件下對鮪魚蒸煮液進行水解作用0-6 h，觀察其水解物對抑制DPP-IV之活性能力。其實驗結果如下：
1. 鮪魚蒸煮液水分含量為93.75%，蛋白質5.44%，而脂質及灰分則占0.23%及1.12%。而總胺基酸分析中，甘胺酸 (Gly) 與組胺酸 (His) 含量最高，其次為丙胺酸 (Ala) 及脯胺酸 (Pro) 等疏水性胺基酸所占比例較高。
2. 於酵素濃度與水解度關係試驗中，發現水解度會隨著水解時間及酵素濃度成正向關係。使用1 mg/mL PR及OR水解6 h 後，其水解度上升至19.12% (PR) 及10.25% (OR)。
3. 在抑制DPP-IV活性測定方面，PR及OR水解物在水解0 h時，抑制率為20.7% 及26.4%，利用1mg/mL酵素水解鮪魚蒸煮液1h後，其水解物 (PRH及ORH) 抑制能力明顯上升至36.2%及36.9% (p<0.05)。但隨著酵素濃度增加及水解時間延長，抑制率並沒有顯著增加，因此兩種酵素均選用水解1h且酵素濃度1 mg/mL的水解物作為後續分析條件。
4. 根據此結果，將PRH及ORH經凝膠過濾法進行分子量劃分，探討水解物之分子量對抑制能力之影響，2組水解物分別各得5個區分物，其中分子量範圍大約為1422 Da之區分物2 (PRF2及ORF2) 抑制能力顯著高於其他區分物 (p<0.05)，其IC50為PRF2：7.0 mg/mL；ORF2：8.3 mg/mL。
5. 隨後將PRF2及ORF2以RP-HPLC進行分離純化，各組劃分出5個區分物，經冷凍乾燥後，測定其抑制DPP-IV能力。發現區分物c (PRF2c 及ORF2c) 抑制效果較佳，樣品濃度在2 mg/mL，其抑制率分別達19.5% 及16.1%。
6. 最後經MALDI TOF/TOF 分析鑑定PRF2c 及ORF2c 之胺基酸序列，所得到之胜肽分別為：
Pro-Ala-Cys-Gly-Gly-Phe-Try-Ile-Ser-Gly-Arg-Pro-Gly (1304.644 Da)、Cys-Ala-Tyr-Gln-Trp-Gln-Arg-Pro-Val-Asp-Arg-Ile-Arg(1690.879 Da)、Pro-Gly-Val-Gly-Gly-Pro-Leu-Gly-Pro-Ile-Gly-Pro-Cys-Tyr-Glu(1412.733 Da)。
根據以上結果，具有較佳抑制DPP-IV能力之胜肽，可能與疏水性胺基酸較豐富有相關係。而鮪魚蒸煮液之蛋白質水解物具有抑制DPP-IV能力，有利於開發成為降血糖之保健食品，可望提昇水產加工副產物的經濟及減低廢棄物所帶來的環境污染。
Incretin hormones are defined as intestinal hormones released in response to nutrient ingestion, which exert a potent glucose-dependent insulinotropic action. It also suppresses glucagon secretion, slows gastric emptying and reduces appetite. Incretin is beneficial to provide glycemic control while avoiding hypoglycemia and weight gain. The half-life of incretin in the circulation is less than 2 minutes owing to the rapid inactivation by the ubiquitous proteolytic enzyme dipeptidyl peptidase-IV (DPP-IV). DPP-IV inhibitors have been investigated as a new therapy approach for the treatment of type 2 diabetes mellitus.
The aim of this study was to utilize tuna cooking juice by-product as the material, to be hydrolyzed by Protease XXIII (PR) and Orientase (OR) for 0, 1, 2, 4, 6 h. The inhibitory effect of tuna cooking juice hydrolysates against DPP-IV activity was investigated, and the sequences of the DPP-IV inhibitory peptides were identified. The results were shown as follows:
1. The moisture, protein, lipid and ash contents of tuna cooking juice are 93.75, 5.44, 0.23 and 1.12%, respectively. In tuna cooking juice, glycine and histidine are the major constituted amino acids, and alanine and proline are the most abundant hydrophobic amino acids.
2. The degree of hydrolysis (DH) of tuna cooking juice with PR and OR at the enzyme concentration of 1 and 2 mg/mL increased dramatically during the first 60 min and then increased gradually thereafter. DHs of the hydrolysates obtained after 6 h hydrolysis by PR and OR were 23.5% and 19.4%, respectively.
3. The 1 h PR and OR hydrolysates had significantly (p<0.05) higher DPP-IV inhibitory activity than 0h hydrolysates, and their DPP-IV inhibitory activities were 36.2% and 36.9%, respectively.
4. After fractionation on a Sephadex G-25 gel filtration, PRH and ORH both had five (F1-5) major fractions. PRF2 and ORF2 showed the highest DPP-IV inhibitory activities of 39.5% and 38.8%, respectively among all the fractions, and their IC50 values were 7.0 mg/mL and 8.3 mg/mL.
5. Identification of the DPP-IV inhibitory peptides were accomplished with RP-HPLC, and five fractions (a-e) were obtained for both PRF2 and ORF2. The PRF2c and ORF2c showed the highest DPP-IV inhibitory activities of 19.5% and 14.8% at the concentration of 2 mg of dried extract/mL, respectively, as compared to the other fractions.
6. Finally, the amino acid sequences of the DPP-IV inhibitory peptides isolated from the PRF2c and ORF2c were identified:
Pro-Ala-Cys-Gly-Gly-Phe-Try-Ile-Ser-Gly-Arg-Pro-Gly (1304.644 Da)、Cys-Ala-Tyr-Gln-Trp-Gln-Arg-Pro-Val-Asp-Arg-Ile-Arg (1690.879 Da)、Pro-Gly-Val-Gly-Gly-Pro-Leu-Gly-Pro-Ile-Gly-Pro-Cys-Tyr-Glu (1412.733 Da)。
The results showed that the peptides majorly constituted by hydrophobic amino acids and having the proline or alanine as the penultimate N-terminal residue possessed great DPP-IV inhibitory activity. Tuna cooking juice could be the potential source of DPP-IV inhibitor that may be used in the treatment of type 2 diabetes. |
