Online - 2455-3891 Vol 10, Issue 3, 2017 Print - 0974-2441 Research Article ANGIOTENSIN CONVERTING ENZYME INHIBITORY ACTIVITY OF MELINJO (GNETUM GNEMON L.) SEED EXTRACTS AND MOLECULAR DOCKING OF ITS STILBENE CONSTITUENTS ABDUL MUN’IM1*, MUHAMMAD ASHAR MUNADHIL1, NURAINI PUSPITASARI1, AZMINAH2, ARRY YANUAR2 1Department of Pharmacognosy-Phytochemistry, Faculty of Pharmacy, Universitas Indonesia, Depok 16424, Indonesia. 2Department of Biomedical Computation, Faculty of Pharmacy, Universitas Indonesia, Depok 16424, Indonesia. Email: [email protected] Received: 10 November 2016, Revised and Accepted: 30 November 2016 ABSTRACT Objectives: To evaluate the angiotensin converting enzyme (ACE) inhibitory activity of melinjo (Gnetum gnemon) seed extract and to study molecular docking of stilbene contained in melinjo seeds. Methods: Melinjo seed powders were extracted with n-hexane, dichloromethane, ethyl acetate, methanol, and water successively. The extracts were evaluated ACE inhibitory activities using ACE kit-Wist and the phenolic content using Folin–Ciocalteu method. The extract demonstrated the highest ACE inhibitory activity was subjected to liquid chromatography-mass spectrometry (LC-MS) to know its stilbene constituent. The stilbene constituents in melinjo seed were performed molecular docking using AutoDock Vina, and ligand-receptor Interactions were processed using Ligand Scout. Results: The ethyl acetate extract demonstrated the highest ACE inhibition activity with inhibitory concentration 50% value of 9.77 × 10 −8 μg/mL inhibitor),and the highest as in silicototal phenolicmolecular content docking (575.9 studies mg demonstrated gallic acid equivalent/g). that they fit Ultra-performance into the lisinopril receptors.LC-MS analysis of ethyl acetate extract has detected the existency of resveratrol, gnetin C, ε-viniferin, and gnemonoside A/B. These compounds displayed similar physiochemical properties to lisinopril (ACE Conclusion: In vitro analysis ethyl acetate extract from melinjo seeds demonstrated the highest ACE inhibitory activity. Molecular docking analysis indicated that resveratrol dimers, gnetin C and gnemonoside A can be considered ACE inhibitor. Keywords: Angiotensin converting enzyme inhibitor, Gnetum gnemon, Melinjo, Total phenolic, Antihypertension, Molecular docking. © 2017 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (http://creativecommons. org/licenses/by/4. 0/) DOI: http://dx.doi.org/10.22159/ajpcr.2017.v10i3.16108 INTRODUCTION the angiotensin converting enzyme (ACE) inhibitory activity test and determined total phenolic content of melinjo seed extracts. Indonesia abounds with natural resources including 30,000 plant species of 40,000 species in worldwide, while the 940 of them are METHODS medicinal plants [1]. However, only a few of them that had been studied and proved benefits also the mechanism of action scientifically. Materials Medicinal plants and vegetables provide stilbene with the interesting biological activity including apoptosis induction of colon cancer [2] Province and authenticated by Plant Conservation Center of Indonesia and blood glucose reduction [3]. One of stilbene derivates, resveratrol ScienceMelinjo Agencies,seeds were Indonesia. obtained Thefrom voucher Melinjo specimen Production was Center, deposited Banten in which was known to provide anti-aging activity [4], antidiabetic, Herbarium Laboratory of Pharmacognosy-Phytochemistry, Faculty of cardioprotective, anticancer [5], antioxidant [6], anti-inflammatory, and antithrombotic [7] so that many studies had been conducted to find the acid, methanol, dichloromethane, n-hexane, ethyl acetate, sodium other possible potential effect which could benefit human life. chloride,Pharmacy, and Universitas dimethyl Indonesia.sulfoxide (DMSO) ACE kit were (Dojindo, purchased and Japan), from Merck, gallic Germany. Genus Gnetum (Gnetaceae family) has been known to contains stilbene, Preparation of extract Gnetum gnemon L. or locally known as Melinjo, which widely cultivated Melinjo powder (1 kg) was refluxed with n-hexane and then filtered. insuch Southeast as resveratrol, Asia. Thein high fruit, concentration seed, leave, [3,8,9].and flowers One of ofthose melinjo plants can is The same procedure was repeated 2 times. The organic solvent was be consumed. This plant is the staple food in some places. Melinjo combined, and evaporated using rotary vacuum evaporator at 40°C, and is rich-in resveratrol, so very helpful in health aspect because the dried using oven vacuum to get hexane extract. The same procedure effect was associated with protection of cardiovascular disease, and was applied using dichloromethane, ethyl acetate, methanol and water, some of cancers such as breast cancer, prostate cancer, and colorectal successively to get dichloromethane, ethyl acetate and methanol, cancer [10-13]. excluded water extract. The water solution was dried using freeze dryer to get water extract. The part of melinjo plant which has health benefits were the seed, leave, and root parts. The main component of melinjo seed is stilbene Extractweight %Extract yield=×100 compound consisting gnemonoside A, gnemonoside C, gnemonoside Herbweight D, gnetin C, gnetin L, and resveratrol [14]. Stilbene and flavonoid are polyphenolic compounds. The availability of polyphenolic compound in In vitro ACE inhibition activity test this melinjo thus indicated that melinjo has many biological activities Dry extracts were dissolved in DMSO and distilled water and made which benefit on human body. According to that reason, we evaluated in a final concentration of 100 µg/ml. Each extract was subjected Mun’im et al. Asian J Pharm Clin Res, Vol 10, Issue 3, 2017, 243-248 to ACE inhibitory assay using Dojindo ACE Kit-WST test kit on pharmacophoric feature points instead of chemical structure. protocol [15-17]. This assay used 3-hydroxybutyrate glycyl RESULTS AND DISCUSSION (Dojindo Laboratories, Japan) as instructed in the manufacturer’s The result of authentification of the sample stated that the sample was inhibitory. The absorbance value of the assay reaction was measured glycylglycine (3HB-GGG) as the substrate for screening of the ACE G. gnemon L. Gnetaceae family. The sample was sorted to eliminate contaminant. First, we did wet sortation to separate the unwanted (Kimia Farma Ltd, Indonesia) was used as a positive control in this plants part. After that, we washed to clean the plant part from the dirt. study.at 450 nm using microplate reader (Thermo Scientific, USA). Captopril The cleaned part then cut into small parts to ease and fasten the drying process. The next process was drying process. The chosen drying process was preventing direct sun exposure in the drying process so ACE inhibitory activity was measuredAAblanks1− usingam plthise following formula: that hopefully the secondary metabolite contained in the study plant ACEinhibitionactivity(%)= will not be destroyed [20]. Then, we did dry sortation to separate the AA− blankb12lank herb from other unwanted things. The red fruit (ripe) was separated from the seed skin in brown which then separated again to obtain the yellow seed. Ablank 1 = Absorption values of positive controls (100% ACE), AWith: = Absorption values of the reagents (without ACE), blank 2 The last step of study plant was the minimizing herb size using A = Absorption values of the extract or captopril. sample instruments that appropriate for the herb characteristics. The minimizing process of herb was conducted using grinder, to obtain Inhibitory concentration 50% (IC ) measurement could be found using 50 the higher extraction area so that it provided maximum process. various concentrations of samples linear regression equation, sample The filtering process was conducted using sieve paper. The too small concentrations as x-axis and the inhibition percentage as the y-axis so herb powder (soft powder) was not wanted because the soft powder values using this following 50 could be released in the filtering process. While the too big size also that using: y = a + bx we could measure IC unwanted because this will disturb the maximum extraction process. 50 a formula:− The herb then saved in a dry and protected container from the sunlight = b melinjo. Determination of total phenolic content to maintain the quality [21]. We obtained 0.89 kg herb from 4 kg wet Total phenolic content was measured by Folin–Ciocalteu method Melinjo dry powder (1 kg) was extracted using reflux method using gradual dissolution. The chosen method was extracted using reflux in 25 ml of methanol, 0.5 ml aliquot of diluted was mixed with method because this method was fast and had been widely used [22]. 2.5referring ml of to10% the Folin–Ciocalteu Borkataky [18]. reagent, The extract vortexed (25 andmg) incubatedwas diluted at The gradual extraction technique used to maximize the extraction 25°C. After 2 minutes, 2 ml of 7.5% Na2CO3 was added. The mixture process, whereas the compounds will be extracted based on the polarity was vortex and incubated for 60 minutes at 25°C. The absorbance properties. of the mixture was measured at 765 nm with a ultraviolet-visible spectrophotometer. Gallic acid was used as a reference, and the In the extraction process, we obtained methanol extract provided results were expressed as mg of gallic acid equivalents (GAE) per g the highest yield number with 6.37% followed by water extract with 3.90%, n-hexane, ethyl acetate, and dichloromethane with the times repetition (triplo).