ACE Inhibitory Activity, Total Phenolic and Flavonoid Content of Watercress (Nasturtium officinale R. Br.) Extract

Introduction: Hypertension is the main risk factor for cardiovascular disease. There are many developed antihypertension drugs, one of them is focusing in ACE (Angiotensin Converting Enzyme) inhibition activity. ACE inhibition activity known can decrease vasoconstriction effect and also can decrease bradykinin degradation (vasodilator) by creating NO (nitric oxide). Methods: In this study, we conducted an in vitro ACE inhibition activity test which was obtained from watercress on 70% ethanolic extract and each fraction (n-hexane, ethyl acetate, and n-butanol). Results: Results of the study showed that ethanolic extract of watercress had ACE activity with IC50 value was 19.05 μg/mL and the highest IC50 of each fraction is ethyl acetate with IC50 value was 2,303 μg/ mL. n-butanol fraction had the highest total phenolic content with 15.798 mg GAE/g of the extract, while the highest total flavonoid content was obtained on ethyl acetate fraction with 82.847 mg QE/g of the extract. Conclusion: The results suggest that Watercress (Nasturtium officinale R. Br.) possess ACE inhibitory activity.


INTRODUCTION
Hypertension is the biggest challenge in Indonesia. Based on Riset Kesehatan Dasar data on 2013, there was quite high hypertension prevalence, which was shown as 25.8%. Every year, hypertension has caused the death of 9.4 million people. WHO predicted that in 2025 there will be around 29% of world population that suffered from hypertension and will increase if they don't follow a healthy lifestyle. The highest percentage of hypertension now obtained from development countries. 1 Based on clinical studies, antihypertension drugs such as Angiotensin Converting Enzyme inhibitor (ACEi), Angiotensin Receptor Blocker (ARB), betablocker (BB), Calcium Channel Blocker (CCB), and thiazide diuretics can lower hypertension complications without affecting the target organs. 2 The raise of blood pressure can be caused by the lowering effect of peripheral pressure. NO (Nitric Oxide) is a vasodilator agent works by lowering the peripheral pressure. ACE (Angiotensin Converting Enzyme) inhibition works by inhibiting the production of Angiotensin II and increase bradykinin level. Bradykinin induces receptor on endothelial cells and causing NOS3 (Nitrate Oxide Synthase 3) activation, NOS3 (endothelial) can convert arginine into NO and produce vasodilatation effect. Watercress (Nasturtium officinale R. Br.) is a widely consumed plant by the population. This plant usually used as food, while many people also use them as traditional medicine. Watercress (N. officinale R. Br.) known has ACE inhibition activity, this is shown with IC50 value of the methanolic extract of N. officinale R. Br. which is 15.40 μg/mL and contained alkaloid, saponin, anthraquinone, terpenoid, and tannin compounds. 3 On this study, we conducted ACE inhibition activity test which was obtained from watercress (Nasturtium officinale R. Br.), continued with each fraction (n-hexane, ethyl acetate, and n-butanol) and also a measurement of total phenolic content and total flavonoid content.

MATERIALS AND METHODS
This study was conducted in Phytochemical Laboratory and Quantitative Analysis of Pharmaceutical Chemistry of the Universitas Indonesia, Depok. Work procedures done were material preparations, extractions, fractionations, ACE inhibition percentage measurements and IC50 test from the extract and also total phenolic and flavonoid content measurements on watercress (Nasturtium officinale R. Br.) fractions.

Material Preparations
Plant determination was conducted to confirm that we used the right plant, such as watercress (Nasturtium officinale R. Br.). Plant identification result showed that sample was in Brassicaceae Family, Nasturtium officinale R. Br. Species.

Botanical Extraction
The method used in extraction process in this study was maceration with ethanol 70% as the solvent, aiming to separate some secondary metabolites in the botanical powder. Maceration method was chosen because the device was simple and safe to use for thermolabile compounds because this method does not need heat. er the phenolic ion contained in the sample solutions. Phenolic concentration data from each watercress (Nasturtium officinale R. Br.) fraction shown in the Table 3 Maceration was conducted by soaking the botanical powder which had been put in a cloth soaked with 2.5 L ethanol 70% solvent for 2x24 hours and stirred f o r every one hour until the first six hours reached, remaceration was conducted to ensure that the chemical compounds contained in the study plant wsere separated completely. Remaceration process was conducted until 7 times, then the macerate was collected and separated using filter papers to prevent filtrate to be mixed with the lees, then the filtrate was evaporated using evaporator device on 55 o C with 50 rpm rate until obtained a condensed extract. After evaporation process, we obtained a condensed extract of watercress plant (Nasturtium officinale R. Br.) as much as 124.2 grams, then we measured the dissolved compounds in the solvent which was used to obtain the yield. The yield of watercress condensed extract (Nasturtium officinale R. Br.) was 24.84%.

Fractionation
In this study, fractionation was conducted with 50.13 grams extract on n-hexane (non-polar), ethyl acetate (semi-polar), and n-butanol (polar) as the solvent. The raised polarity properties of the solvent used wsere done so that the compounds in the botanical can be extracted and separated based on the polarity properties of the compounds. Each fraction yields obtained from n-hexane, ethyl acetate and n-butanol were 3.591, 3.271, and 7.341%. The final yield of the fractionation process was in the water phase.

ACE Inhibition Activity Test
The in vitro ACE inhibition activity test of watercress (Nasturtium officinale R. Br.) was measured using ACE Kit-WST (Dojindo, Japan) because of the fast, accurate, and specific process. The main principle of the study using this method is measuring 3HB (3-hydroxybutyrate) absorption obtained from the ACE catalyzation process towards 2HB-GGG and WST-1 formazan substrates. Results of the study toward the inhibition value of watercress were 94.31%, then on the sample extract, we measured the IC 50 values in five various final concentration, such as 100.5, 50.25, 25.13, 12.56, and 6.28 μg/mL. IC 50 value of the ethanolic extract of watercress obtained was 19.05 μg/mL. The result could be found below. Data of ACE inhibition activity in 70% ethanolic extract cand be found in Table 1 This study was compared with captopril as the standard of ACE inhibition activity and the percentage value of ACE activity obtained was 34.34% on 5x10-8 ppm, this showed that watercress (Nasturtium officinale R. Br.) provide ACE inhibition activity, but lower than captopril because in the botanical powder still contained other chemical compounds, while captopril is a pure compound. Then ACE inhibition activity was test in each fractions (n-hexane, ethyl acetate, n-butanol) and the result was 71,31% (100 µg/mL); 75,74% (50 µg/mL); 78,78% (100 µg/mL) respectively. The highest inhibition value obtained in ethyl acetate fraction with IC 50 was 2,303 μg/mL. IC 50 ethyl acetate could be found in Table 2. IC 50 curve of ethyl acetate fraction of Watercress can be found in Figure  1 and data of ACE inhibition activity in ethyl acetate fraction can be found in Table 2.

Total Phenolic Content Measurement
Total phenolic content of watercress fractions was measured using Folin-Ciocalteu method. Folin-Ciocalteu reagent was used as color complex because a reaction between gallic acid and Folin-Ciocalteu can form a stable complex with blue colored. The denser the colour means the high-     (Nasturtium officinale R. Br.) in ethyl acetate fraction provide Angiotensin Converting Enzyme (ACE) inhibition with IC 50 value was 2.303 μg/mL. Watercress (Nasturtium officinale R. Br.) ethyl acetate fraction had the highest antioxidant activity with EC 50 value was 18.816 μg/mL. Watercress (Nasturtium officinale R. Br.) n-butanol fraction had total phenolic content value on 3.624 mg QE/g extract and ethyl acetate fraction with the highest total flavonoid content with 2.710 mg QE/g extract.

ACKNOWLEDGEMENT
This study was supported by The Directorate of Research and Community Engagement Universitas Indonesia via Hibah PITTA 2016.