Aktivitas Anti-Alzheimer Ekstrak Rimpang Temulawak Melalui Inhibisi Enzim Asetilkolinesterase dan Pendekatan Molecular Docking

Abstract

Penyakit Alzheimer (AD) merupakan penyebab utama demensia yang ditandai oleh penurunan fungsi memori dan kognitif secara progresif. Salah satu mekanisme utamanya adalah degradasi asetilkolin yang berlebihan oleh enzim asetilkolinesterase (AChE) yang mengganggu transmisi kolinergik serta berkontribusi terhadap pembentukan plak ß-amiloid dan serabut neurofibrilar. Rimpang temulawak diketahui mengandung senyawa bioaktif yang berpotensi menghambat aktivitas AChE secara in vitro dan in silico. Penelitian ini bertujuan menganalisis dan mengevaluasi potensi ekstrak EtOH 70% rimpang temulawak sebagai kandidat penghambat AChE melalui pendekatan terintegrasi kimia, biologi, dan komputasi yang terintegrasi. Metode penelitian meliputi analisis kadar air simplisia dengan metode gravimetri serta perhitungan rendemen ekstrak hasil ekstraksi maserasi-remaserasi menggunakan EtOH 70%. Kadar senyawa penciri, yaitu kurkuminoid (CUR, DMC, BDMC) dan xantorizol (XNT) dianalisis menggunakan HPLC. Uji penghambatan aktivitas AChE dilakukan secara in vitro untuk menentukan nilai IC50 ekstrak. Profil metabolit non-targeted diidentifikasi menggunakan UHPLC Vanquish Q Exactive Plus Orbitrap HRMS. Senyawa yang teridentifikasi dievaluasi secara in silico melalui analisis residu penting AChE, preparasi reseptor dan ligan uji, penambatan molekuler, prediksi ADMET dan drug-likeness, serta simulasi dinamika molekular. Donepezil sebagai kontrol positif. Hasil menunjukkan kadar air simplisia sebesar 8,55% dan rendemen ekstrak sebesar 25,37%. Kadar kurkuminoid sebesar 44,54 mg/g ekstrak (CUR); 17,73 mg/g ekstrak (DMC), dan 0,96 mg/g ekstrak (BDMC), sedangkan kadar xantorizol sebesar 97,57 mg/g ekstrak. Nilai IC50 donepezil dan ekstrak masing-masing sebesar 0,03±0,00 µg/mL dan 46,63±0,17 µg/mL. Identifikasi senyawa nontargeted didominasi oleh senyawa golongan seskuiterpen dan kurkuminoid dari 43 senyawa yang teridentifikasi. Evaluasi rongga pengikatan ligan pada AChE menunjukkan adanya gorge yang dalam dan sempit yang terdiri atas situs katalitik dan situs anionik perifer sebagai area penghambatan. Penambatan molekular menunjukkan BDMC dan CYC memiliki energi ikatan lebih besar dibandingkan donepezil, sedangkan DMC, CUR, dan XNT mendekati energi ikatan donepezil. Prediksi ADMET dan drug-likeness menunjukkan senyawa xantorizol memiliki profil bioavailabilitas dan keamanan paling baik dibandingkan kontrol. Simulasi dinamika molekuler mengonfirmasi kestabilan interaksi kompleks AChE-XNT selama simulasi 5 ns, yang didukung oleh parameter RMSD, RMSF, Rg, SASA, dan MM-PBSA. Simpulan penelitian ini yaitu ekstrak EtOH 70% rimpang temulawak berpotensi sebagai sumber senyawa anti-Alzheimer melalui mekanisme penghambatan enzim AChE melalui integrasi in vitro dan in silico.

Alzheimer's disease (AD) is the leading cause of dementia, characterized by progressive decline in memory and cognitive function. One of its main mechanisms is excessive degradation of acetylcholine by the enzyme acetylcholinesterase (AChE), which disrupts cholinergic transmission and contributes to the formation of ß-amyloid plaques and neurofibrillary tangles. Curcuma rhizome is known to contain bioactive compounds that have the potential to inhibit AChE activity in vitro and in silico. This study aims to analyze and evaluate the potential of 70% ethanol extract of curcuma rhizome as a candidate AChE inhibitor through an integrated approach of chemistry, biology, and computation. The research methods included analyzing the moisture content of the crude drug using the gravimetric method and calculating the yield of the extract obtained from maceration-remaceration using 70% ethanol. The content of characteristic compounds, namely curcuminoids (CUR, DMC, BDMC) and xanthorrhizol (XNT), was analyzed using HPLC. The AChE activity inhibition test was conducted in vitro to determine the IC50 value of the extract. The untargeted metabolite profile was identified using UHPLC Vanquish Q Exactive Plus Orbitrap HRMS. The identified compounds were further evaluated in silico through AChE key residue analysis, receptor and test ligand preparation, molecular docking, ADMET and drug-likeness prediction, and molecular dynamics simulation. Donepezil was used as a positive control. The results showed that the moisture content of the crude drug was 8.55% and the extract yield was 25.37%. The curcuminoid content was 44.54 mg/g extract (CUR); 17.73 mg/g extract (DMC), and 0.96 mg/g extract (BDMC), respectively, while the xanthorrizol content was 97.57 mg/g. The IC50 values for donepezil and the extract were 0.03±0.00 µg/mL and 46.63±0.17 µg/mL, respectively. Identification of non-targeted compounds was dominated by sesquiterpenes and curcuminoids among the 43 identified compounds. Evaluation of the ligand-binding pocket in AChE revealed a deep, narrow gorge consisting of a catalytic site and a peripheral anionic site as the inhibition area. Molecular docking showed that BDMC and CYC had higher binding energies compared to donepezil, while DMC, CUR, and XNT were close to donepezil’s binding energy. ADMET and drug likeness predictions indicate that the xantorizol compound has the best bioavailability and safety profile compared to the control. Molecular dynamics simulations confirmed the stability of the AChE-XNT complex interactions during a 5 ns simulation, supported by the RMSD, RMSF, Rg, SASA, and MM-PBSA parameters. The conclusion of this study is that a 70% EtOH extract of temulawak rhizome has potential as a source of anti-Alzheimer’s compounds through the mechanism of AChE inhibition, as demonstrated by in vitro and in silico analyses.