| Summary: | 碩士 === 國立屏東科技大學 === 生物科技系所 === 105 === Hypertension is a chronic disease, and now becomes the most common disease. Although some chemically synthesized antihypertensive drugs have been developed, they are usually accompanied by side effects. It is generally believed that the hypotensive active peptides obtained from natural sources have lower side effects than chemically synthesized drugs.
In this study, Cucurbita moschata seed proteins were extracted and hydrolyzed using thermolysin as digestion enzyme. Small peptides were obtained using super ultrafiltration by means of molecular weight cut-off membranes (3kDa cut-off). The small peptides were fractionated using reversed-phase high performance liquid chromatography (RP-HPLC) and the angiotensin-converting enzyme (ACE) inhibitory activities of the resulting fractions were examined by in vitro ACE inhibitory assay. Peptide identification for the most active fraction was performed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) coupled with database-assisted sequencing, IR-8 was identified from the fraction with the most effective ACE inhibition and the identity and activity of IR-8 were further confirmed by synthetic peptide.
The results showed that the IC50 value of IR-8 was 14.2±1.6 μM. Inhibition kinetics showed that IR-8 was a competitive inhibitor. Furthermore, the inhibition type of IR-8 was determined using a pre-incubation experiment. The results showed that IR-8 had a significant decrease in IC50 after pre-incubated with ACE, which suggested that IR-8 was a prodrug. In this study, we successfully uncovered the potent ACE inhibitory peptide IR-8 from the thermolysin hydrolysate of pumpkin seeds. The antihypertensive effect of IR-8, as well as thermolysin hydrolysate of pumpkin seeds will be explored in the future.
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