Sweet Potato Storage Root Thioredoxin h2 and Their Peptic Hydrolysates Exhibited Angiotensin Converting Enzyme Inhibitory Activity in vitro

甘藷塊根中硫氧化還原蛋白h2及其合成之胜肽具有血管收縮素轉化酶抑制活性

黃冠中(Guan-Jhong Huang) ； 陳顯榮(Hsien-Jung Chen) ； 北中進(Kitanaka Susumu) ； 吳金濱(Jin-Bin Wu) ； 侯文琪(Wen-Chi Hou) ； 吳介信(Chieh-Hsi Wu) ； 許明志(Ming-Jyh Sheu) ； 黃世勳(Shyh-Shyun Huang) ； 林耀輝(Yaw-Huei Lin)

《Botanical Studies》 52卷1期 (2011/01) Pp. 15-22

摘要

在大腸桿菌(M15)中大量表現重組蛋白質硫氧化還原蛋白h2(Trx h2)，利用鎳離子螯合之親和性管柱純化。Trx h2經SDS-PAGE分析其分子量約為14kDa。由於Trx h2具有抗氧化活性，去氫抗壞血酸還原酶，單去氫抗壞血酸還原酶的活性。Trx h2以(N-(3-[2-furyl] acryloyl)-Phe-Gly-Gly) (FAPGG)為受質，利用分光光度計的方法分析抑制血管收縮素轉化酶(angiotensin converting enzyme, ACE)的能力，其效果隨劑量增加而增加（50到200μg/mL TI，分別抑制31.9-65.9%血管收縮素轉化酶活性）。Trx h2對於血管收縮素轉化酶之50%抑制濃度(IC50)為151.8μg/mL，對照組Captopril為10nM (868ng/mL)。另外利用螢光silica TLC偵測FAPGG及其水解產物FAP，結果也顯示Trx h2對於血管收縮素轉化酶有抑制的效果。Trx h2對於血管收縮素轉化酶是屬於混合型抑制。而當加入200μg/mL Trx h2時，V(下標 max)和K(下標 m)分別為0.010ΔA/min和0.125mM；而未入Trx h2時，V(下標 max)和K(下標 m)分別為0.0096ΔA/min和0.495mM利用胰蛋白酶以不同時間水解Trx h2時，發現反應16小時時其血管收縮素轉化酶活性有抑制的效果可以從52%(0h)增加到72%(16h)。由結果可知小分子的胜肽會隨著水解時間增加且血管收縮素轉化酶活性抑制也有增加，但水解時間超過16h時，血管收縮素轉化酶活性抑制會降低，可能是由於一些胜肽的結構被破壞。利用電腦模擬胰蛋白酶水解Trx h2的結果，四種人工合成具有抑制血管收縮素轉化酶活性胜肽：EVPK, VVGAK, FTDVDFIK和MMEPMVK，測定其IC50為1.73±0.24，1.14±0.13，0.42±0.02和1.03±0.58mM。結果發現FTDVDFIK具有很好的抑制血管收縮素轉化酶活性。當人們食用甘藷塊根時，Trx h2及其胜肽也許對於高血壓和其他疾病的控制是有益的。

關鍵字

甘藷 ； 血管收縮素轉化酶 ； 硫氧化還原蛋白h2 ； 水解胜肽

並列摘要

Recombinant thioredoxin h (Trx h2) overproduced in E. coli (M15) was purified by Ni(superscript 2+)-chelate affinity chromatography as previously reported (Huang et al., 2004a). The molecular mass of Trx h2 was ca. 14 kDa as determined by SDS (sodium dodecyl sulfate)-PAGE (polyacrylamide gel electrophoresis). Trx h2 had antioxidant (Huang et al., 2004b), dehydroascorbate reductase, and monodehydroascorbate reductase activities (Huang et al., 2008a). Trx h2 was shown by spectrophotometric methods to inhibit angiotensin converting enzyme (ACE) in a dose-dependent manner (50-200 μg/mL, with 31.9~65.9% inhibition) using N-[3-(2-furyl) acryloyl]-Phe-Gly-Gly (FAPGG) as a substrate. A 50% inhibition (IC50) of ACE activity required 151.8 μg/mL of Trx h2 and 10 nM (868 ng/mL) of Captopril. TLC data also showed Trx h2 as an ACE inhibitor. Trx h2 acted as a mixed type inhibitor against ACE using FAPGG as a substrate. When 200 μg/mL Trx h2 were added, Vmax and Km were, respectively, 0.010 ΔA/min and 0.125 mM; without Trx h2 they were 0.0096 ΔA/min and 0.495 mM. Trypsin was used for Trx h2 hydrolysis over different time periods. ACE inhibitory activity was found to increase from 52% to about 72% after 16 h of hydrolysis. The results suggested that the ACE inhibitory capacity of small peptides increased through trypsin hydrolysis of Trx h2 up to 16 h and then decreased, which may have been due to the disappearance of some active ingredients. Four peptides, namely EVPK, VVGAK, FTDVDFIK and MMEPMVK, were synthesized based on the simulated trypsin digestion of Trx h2 and then tested for ACE inhibitory activity. The IC50 values of individual peptides were 1.73 ± 0.24, 1.14 ± 0.13, 0.42 ± 0.02, and 1.03 ± 0.58 mM, respectively, suggesting that FTDVDFIK might be the main active site of ACE inhibition. The results for Trx h2 and its hydrolysates might mean that consumption of sweet potato storage roots can aid in the control of hypertension and other diseases.

並列關鍵字

Angiotensin converting enzyme ace ； Hydrolysis peptides ； Sweet potato ； Thioredoxin h2.