Sweet Potato Storage Root Defensin and Its Tryptic Hydrolysates Exhibited Angiotensin Converting Enzyme Inhibitory Activity in vitro

甘藷塊根中防禦素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) ； 陸德齡(Te-Ling Lu) ； 邱傳淞(Chuan-Sung Chiu) ； 陳顯榮(Hsien-Jung Chen) ； 吳介信(Chieh-Hsi Wu) ； 林穎志(Ying-Chin Lin) ； 謝文聰(Wen-Tsong Hsieh) ； 廖容君(Jung-Chun Liao) ； 許明志(Ming-Jyh Sheu) ； 林耀輝(Yaw-Huei Lin)

《Botanical Studies》 52卷3期 (2011/07) Pp. 257-264

摘要

在大腸桿菌(M15)中大量表現甘藷塊根之重組蛋白質防禦素(SPD1)，利用鎳離子螯合之親和性管柱純化。SPD1經SDS-PAGE分析其分子量約爲8,600Da。由以前的研究發現SPD1具有抗微生物活性，去氫抗壞血酸還原酶，單去氫抗壞血酸還原酶的活性。SPD1以N-[3-(2-furyl)acryloyl]-Phe-Gly-Gly(FAPGG)爲受質，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/mLSPD1，分別抑制27.56~52.58%血管收縮素轉化酶活性）。SPD1對於血管收縮素轉化酶之50%抑制濃度(IC50)爲190.47μg/mL，對照組Captopril爲10nM(868ng/mL)。另外利用螢光silicaTLC偵測FAPGG及其水解產物FAP，結果也顯示SPD1對於血管收縮素轉化酶有抑制的效果。SPD1對於血管收縮素轉化酶是屬於混合型抑制。利用胰蛋白脢以不同時間水解SPD1時，發現反應24小時時其血管收縮素轉化酶活性有抑制的效果可以從52.47%(0h)增加到74.38%(24h)。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24h時，血管收縮素轉化脢活性抑制會降低，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SPD1的結果，得到六種人工合成具有抑制血管收縮素轉化酶活性胜肽：GFR，FK，IMVAEAR，GPCSR，CFCTKPC和MCESASSK，測定其IC50爲94.25±0.32，265.43±1.24，84.12±0.53，61.67±0.36，1.31±0.07和75.93±0.64μM。結果發現CFCTKPC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，SPD1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 ； 防禦素 ； 血管收縮素轉化酶 ； 抑制作用

並列摘要

Sweet potato defensin (SPD1) overproduced in E. coli (M15) was purified by Ni(superscript 2+)-chelate affinity chromatography. The molecular mass of SPD1 is about 8,600 Da determined by SDS (sodium dodecyl sulfate)-PAGE (polyacrylamide gel electrophoresis). Our previous paper showed that SPD1 had antimicrobial, dehydroascorbate reductase and monodehydroascorbate reductase activities. The activity of SPD1 to inhibit angiotensin converting enzyme (ACE) was shown using N-[3-(2-furyl) acryloyl]-Phe-Gly-Gly (FAPGG) as substrate in a dose-dependent manner (27.56~52.58 % inhibition). The 50% inhibition (IC50) of ACE activity required 190.47 μg/mL SPD1 while that of Captopril was 10 nM (868 ng/mL). Thin layer chromatography (TLC) also identified SPD1 as an ACE inhibitor. SPD1 acted as a mixed type inhibitor against ACE using FAPGG as a substrate. When 200 μg/mL SPD1 (10 μg) were added, Vmax and Km were 0.01 ΔA/min and 0.69 mM, respectively; without SPD, 0.03 ΔA/min and 0.42 mM. Trypsin was used for SPD1 hydrolysis and part of the reaction mixture was removed and analyzed at set times. ACE inhibitory activity increased from 52.47% to about 74.38% after 24 h hydrolysis. The results suggested that small peptides increased by trypsin hydrolysis of the SPD1 ACE inhibitory capacity also increased up to 24 h, then decreased, which may be due to the disappearance of some active ingredients. Six peptides, namely GFR, FK, IMVAEAR, GPCSR, CFCTKPC and MCESASSK, were synthesized based on the simulated trypsin digest of SPD1, then tested for ACE inhibitory activity. IC50 values of individual peptides were 94.25±0.32, 265.43±1.24, 84.12±0.53, 61.67±0.36, 1.31±0.07 and 75.93±0.64 μM, respectively, suggesting that CFCTKPC might represent the main domain for the ACE inhibition. The consumption of sweet potatoes may thus help alleviate hypertension and other diseases due to their SPD1 and hydrolysate content.

並列關鍵字

Angiotensin converting enzyme ACE ； Defensin ； Inhibition ； Sweet Potato