Evaluation and validation of angiotensin-converting enzyme inhibitory activity of moringa oleifera, quercetin, and isoquercetin: A comparative study of fluorescence and absorbance measurement methods.

Moringa oleifera, known for its nutritional benefits and biological functions, such as antihypertensive effects, has cultivated in Japan. However, there is limited information available on moringa cultivated in Japan. Cardiovascular diseases, including ischemic heart disease and stroke, cause many deaths worldwide each year, with hypertension being the most significant risk factor. This study investigated the inhibitory activity of angiotensin-converting enzyme (ACE), which is one of the blood pressure elevation mechanisms, aiming to identify the active compounds in moringa leaves cultivated in Kagoshima prefecture, Japan (Kagoshima-moringa). Additionally, we evaluated the potential overestimation of the ACE inhibitory activity when measured using the fluorescence method. The ACE inhibitor compounds in the methanol-rich extract of Kagoshima-moringa were purified, and mass, aglycone, and glycoside analyses suggested isoquercetin as one of the active compounds. ACE inhibitory activity was evaluated using a fluorescent derivatization method with o-phthalaldehyde (OPA). However, the inhibitory effect of isoquercetin may have been overestimated, as the OPA fluorescence measurement indicated that isoquercetin could inhibit fluorescence readings. Therefore, the absorbance method was performed, revealing that captopril demonstrated similar inhibitory activity, whereas Kagoshima-moringa extracts showed low activity. These results indicate that the ACE inhibitory activity of flavonoids, or samples rich in flavonoids, may be overestimated when assessed by the fluorescence method using OPA. Hence, evaluating ACE inhibitor activity requires the careful selection of measurement methods; multiple activity measurement methods should be used if possible. The study underscores the importance of appropriate measurement methods for evaluating ACE inhibitory activity, guiding future research practices.