ISSN: 2785-2997 Available online at www.HEFJournal.org Journal of Human, Earth, and Future Vol. 5, No. 3, September, 2024 348 Investigating Ethanolic Extract from Acehnese Lime (Citrus aurantifolia) Peel as Potential Anti-Hypercholesterolemia Agent Roma Sitio 1, 2 , Muslim Akmal 3* , Marlina Marlina 4 , Gholib Gholib 5 1 Graduate School of Mathematics and Applied Sciences, Universitas Syiah Kuala, Banda Aceh-23111, (Aceh) Indonesia. 2 Department of Nursing, Polytechnic of Health Ministry of Health, Aceh Besar-23231, (Aceh) Indonesia. 3 Laboratory of Histology, Faculty of Veterinary Medicine, Universitas Syiah Kuala, Banda Aceh-23111, (Aceh) Indonesia. 4 Faculty of Nursing, Medical Surgical Department, Universitas Syiah Kuala, Banda Aceh-23111, (Aceh) Indonesia. 5 Laboratory of Physiology, Faculty of Veterinary Medicine, Universitas Syiah Kuala, Banda Aceh-23111, (Aceh) Indonesia. Received 30 March 2024; Revised 28 July 2024; Accepted 07 August 2024; Published 01 September 2024 Abstract Lime peels are rich in flavonoids, alkaloids, phenols, saponins, and tannins, exhibiting antibacterial, antioxidant, anti- inflammatory, anti-hypertensive, and anti-hypercholesterolemia properties. However, the specific active constituents of Acehnese lime peels and their impact on anti-hypercholesterolemia effects remain undisclosed. This present investigation aims to identify the active compounds in the ethanolic extract of locally obtained Acehnese lime ( Citrus aurantifolia (Christm.) Swingle) peels and assess their potential as inhibitors of Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) and 3-hydroxy-3-methylglutaryl-coenzyme-A (HMG Co-A) reductase using in-silico approach. The composition of the ethanolic extract Acehnese lime peel was determined through phytochemical analysis, 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, gas chromatography-mass spectrometry (GC-MS) analysis, and predictions of biological activity, while the biological activities of their compounds were evaluated through molecular docking. Phytochemicals revealed the presence of phenolics, flavonoids, tannins, saponins, and terpenoids in the ethanolic extract of local Aceh lime peel. The total phenolic and flavonoid contents were 29.992 ± 0.274 mg gallic acid equivalents (GAE)/g extract and 5.983 ± 0.017 mg quercetin equivalents (QE)/g extract, respectively. The anti-oxidant activity was notably strong with an IC50 value of 49.51 ppm. GC-MS analysis identified 6-methoxychroman-2-one (27.64%) as the primary component in ethanolic extract Acehnese lime peel, along with neric acid (C13H22O2) known as a regulator of lipid metabolism (Pa: 0.941). In-silico investigations indicated that pterin-6-carboxylic acid (-7.8 kcal/mol) exhibited a higher binding free energy for the PCSK9 receptor compared to simvastatin (-7.6 kcal/mol), whereas the active compound (R)-9-(2,3-dihydroxy-3-methylbutoxy)-4- exhibited the highest binding capacity for HMG Co-A reductase (-6.9 kcal/mol) compared to other compounds. These findings suggest that the ethanolic extract of Acehnese lime peels could serve as an effective inhibitor of PCSK9 and HMG Co-A reductase, highlighting its potential as a novel anti-hypercholesterolemia agent. Keywords: Citrus aurantifolia; Aceh Lime Peel; Anti-Hypercholesterolemia Agent; PCSK9; HMG-Coa Reductase. 1. Introduction Hypercholesterolemia stands as a significant contributor to cardiovascular disease [1], representing a leading cause of mortality worldwide, accounting for approximately 17.9 million deaths annually [2]. Elevated levels of low-density lipoprotein (LDL) and total cholesterol in the bloodstream characterize hypercholesterolemia [3]. Lifestyle and stress * Corresponding author: akmal_kh@usk.ac.id http://dx.doi.org/10. 28991/HEF-2024-05-03-04 ➢ This is an open access article under the CC-BY license (https://creativecommons.org/licenses/by/4.0/). © Authors retain all copyrights.