Penghambatan Enzim Asetilkolinesterase oleh Ekstrak Etanol Kulit Kayu Manis (Cinnamomum burmannii) Studi In Silico dan In Vitro

Abstract

Penyakit Alzheimer (Alzheimer’s disease, AD) merupakan gangguan neurodegeneratif progresif yang ditandai dengan penurunan fungsi kognitif dan disfungsi sistem kolinergik, termasuk berkurangnya neurotransmisi asetilkolin. Penghambatan enzim asetilkolinesterase (AChE) merupakan salah satu strategi terapi simptomatik untuk mempertahankan ketersediaan asetilkolin. Kulit kayu manis (Cinnamomum burmannii) mengandung senyawa bioaktif yang berpotensi menghambat AChE. Penelitian ini bertujuan mengevaluasi potensi ekstrak etanol kulit kayu manis sebagai inhibitor AChE melalui pendekatan in vitro dan in silico. Profil metabolit ekstrak etanol kulit kayu manis dianalisis menggunakan UHPLC-Q-Orbitrap-HRMS. Aktivitas penghambatan AChE secara in vitro dievaluasi menggunakan metode Ellman dengan donepezil sebagai kontrol positif. Pendekatan in silico dilakukan melalui penambatan molekuler terhadap enzim AChE (PDB ID: 6O4W) untuk memprediksi pola interaksi dan kecenderungan pengikatan senyawa kandidat. Kompleks protein–ligan terpilih selanjutnya dianalisis melalui simulasi dinamika molekuler untuk mengevaluasi karakteristik dinamika dan kestabilannya selama simulasi. Sifat fisikokimia dan drug-likeness diprediksi menggunakan SwissADME, sedangkan parameter farmakokinetik dan toksisitas diprediksi menggunakan pkCSM. Analisis profil metabolit berhasil mengidentifikasi 55 senyawa dalam ekstrak etanol kulit kayu manis, dengan kumarin sebagai senyawa dominan berdasarkan luas area kromatogram relatif sebesar 11,29%. Ekstrak etanol kulit kayu manis menurunkan aktivitas AChE dari 189,10 ± 1,00 mU pada konsentrasi 0,1 µg/mL menjadi 106,10 ± 1,00 mU pada konsentrasi 30 µg/mL, disertai peningkatan inhibisi dari 40,93 ± 0,27% menjadi 66,85 ± 0,42%. Donepezil sebagai kontrol positif menghasilkan inhibisi sebesar 95,91 ± 0,31%. Nilai IC50 ekstrak etanol kulit kayu manis sebesar 1,53 ± 0,02 µg/mL, sedangkan donepezil menunjukkan potensi penghambatan yang lebih tinggi dengan nilai IC50 sebesar 0,03 µg/mL. Hasil analisis penambatan molekuler menunjukkan bahwa asam kafeoilshikimat (CSA) memiliki nilai energi ikatan (?G binding) paling negatif dari senyawa alami, yaitu -10,39 kcal/mol, yang mengindikasikan afinitas pengikatan paling kuat serta potensi interaksi dengan residu-residu penting pada situs katalitik dan Peripheral Anionic Site (PAS) AChE. Hidroksisinamaldehida (HCA) dipilih untuk analisis lebih lanjut karena memenuhi aturan Lipinski serta diprediksi memiliki karakteristik absorpsi baik dan profil toksisitas yang relatif menguntungkan. Simulasi dinamika molekuler selama 5 ns pada suhu 310 K menunjukkan bahwa kompleks AChE–HCA mempertahankan kestabilan relatif selama simulasi jangka pendek, meskipun memiliki fluktuasi lebih tinggi dibandingkan kompleks AChE– DNPZ. Simpulan penelitian ini yaitu ekstrak etanol kulit kayu manis menunjukkan potensi penghambatan AChE secara in vitro, sedangkan HCA merupakan kandidat senyawa yang perlu dikaji lebih lanjut melalui pengujian eksperimental.

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder characterized by cognitive decline and cholinergic system dysfunction, including reduced acetylcholine neurotransmission. Acetylcholinesterase (AChE) inhibition is one of the symptomatic therapeutic strategies used to maintain acetylcholine availability. Cinnamon bark (Cinnamomum burmannii) contains bioactive compounds with potential AChE-inhibitory activity. This study aimed to evaluate the potential of cinnamon bark ethanol extract as an AChE inhibitor using combined in vitro and in silico approaches. The metabolite profile of the cinnamon bark ethanol extract was analyzed using UHPLC-Q-Orbitrap-HRMS. Its in vitro AChE-inhibitory activity was evaluated using the Ellman method, with donepezil as the positive control. The in silico investigation involved molecular docking against AChE (PDB ID: 6O4W) to predict the binding interactions and binding tendencies of candidate compounds. Selected protein–ligand complexes were subsequently subjected to molecular dynamics simulations to assess their dynamic behavior and stability throughout the simulation. Physicochemical properties and drug-likeness were predicted using SwissADME, whereas pharmacokinetic and toxicity parameters were predicted using pkCSM. Metabolite profiling successfully identified 55 compounds in the ethanolic extract of cinnamon bark, with coumarin identified as the predominant compound based on its chromatographic peak area of 11.29%. The ethanolic extract of cinnamon bark reduced acetylcholinesterase (AChE) activity from 189.10 ± 1.00 mU at a concentration of 0.1 µg/mL to 106.10 ± 1.00 mU at 30 µg/mL, accompanied by an increase in the percentage of inhibition from 40.93 ± 0.27% to 66.85 ± 0.42%. Donepezil, used as the positive control, produced an inhibition of 95.91 ± 0.31%. The IC50 value of the ethanolic cinnamon bark extract was 1.53 ± 0.02 µg/mL, whereas donepezil exhibited greater inhibitory potency, with an IC50 value of 0.03 µg/mL. Molecular docking analysis showed that caffeoylshikimic acid (CSA) had the most negative binding energy (?G binding) among the natural compounds, at -10.39 kcal/mol, indicating the strongest binding affinity and the potential to interact with key residues within the catalytic site and the Peripheral Anionic Site (PAS) of AChE. Hydroxycinnamaldehyde (HCA) was selected for further analysis because it complied with Lipinski’s rule of five and was predicted to possess favorable absorption characteristics and a relatively favorable toxicity profile. A 5- ns molecular dynamics simulation conducted at 310 K indicated that the AChE– HCA complex maintained relative stability during the short-term simulation, although it exhibited greater fluctuations than the AChE–DNPZ complex. In conclusion, the ethanolic extract of cinnamon bark demonstrated potential AChE inhibitory activity in vitro, while HCA represents a promising candidate compound that warrants further investigation through experimental validation.