Isolasi dan Identifikasi Senyawa Aktif dari Daun Buas-buas (Premna serratifolia) sebagai Inhibitor Alfa Glukosidase

DINI HADIARTI, Dr. Winarto Haryadi, M.Si

2022 | Disertasi | DOKTOR KIMIA

Telah dilakukan kajian senyawa aktif dari daun Buas-buas (Premna serratifolia) sebagai inhibitor alfa glukosidase. Penelitian ini dilatarbelakangi tradisi masyarakat Kalimantan Barat yang mengkonsumsi daun P. serratifolia sebagai tonik, mengobati sakit kepala, sakit perut, diabetes, hipertensi, kesulitan bernafas, malaria, dan meningkatkan produksi ASI. Ekstrak daun P. serratifolia dapat menginhibisi alfa glukosidase untuk menurunkan kadar glukosa dalam darah. Karena upaya untuk mengisolasi metabolit sekunder dari P. serratifolia sebagai inhibitor alfa glukosidase belum dilakukan, maka diperlukan kajian untuk mengetahui senyawa yang bertanggung jawab dalam aktivitas tersebut yang terbukti secara in vitro maupun in silico. Daun P. serratifolia dikeringkan, dihaluskan, dan dimaserasi dengan etanol (p.a) selama 4 hari. Ekstrak yang diperoleh selanjutnya difraksinasi dengan heksana, etil asetat, dan air. Fraksi-fraksi yang diperoleh kemudian ditentukan aktivitas inhibisi alfa glukosidase, kandungan fitokimia, total kadar fenolik (TPC), flavonoid (TFC), dan steroid (TSC), serta dianalisis dengan spektroskopi Fourier Transform Infrared (FTIR). Pengaruh TPC, TFC, TSC, dan gugus fungsi terhadap inhibisi alfa glukosidase ditentukan dengan Principal Component Analysis (PCA) dan regresi Partial Least Square (PLS). Kromatografi kolom dan Preparative High Pressure Liquid Chromatography (HPLC preparatif) digunakan untuk mengisolasi senyawa dari fraksi dengan inhibisi alfa glukosidase terbaik. Senyawa aktif dari isolat diidentifikasi dengan menggunakan Ultra High Performance Liquid Chromatography-Q Exactive hybrid quadrupole-Orbitrap High Resolution Mass Spectrometry (UHPLC-Q-Orbitrap HRMS). Interaksi alfa glukosidase dan senyawa aktif dikaji dengan molecular docking menggunakan protein N-terminal maltaseglucoamylase (Kode PDB: 2QMJ), C-terminal maltase-glucoamylase (Kode PDB: 3TOP), dan isomaltase (Kode PDB: 3A4A). Skrining fitokimia terhadap ekstrak etanol, fraksi heksana, etil asetat, dan air menunjukkan terdapat flavonoid, fenolik, dan saponin (steroid). Hasil uji in vitro memperlihatkan fraksi etil asetat mempunyai aktivitas inhibisi terbaik dengan IC50 yaitu 10900 ppm. PCA dan PLS menunjukkan TFC dan gugus -C=O mempunyai koefisien standardisasi tertinggi sehingga mempunyai kontribusi terbesar untuk menghambat alfa glukosidase. Uji inhibisi alfa glukosidase terhadap subfraksi etil asetat diperoleh subfraksi F12-F14 dengan IC50 terkecil yaitu < 2000 ppm. Analisis UHPLC-Q-Orbitrap HRMS menunjukkan 9 flavonoid yang berhasil teridentifikasi yaitu centaureidin, chyrsin, glycitein, tricin, pectolinaringenin, 3,5,4'-trimetoksi-6,7-metilendioksiflavon, kaempferide, syringetin, dan casticin. Satu dari 9 flavonoid yang berhasil teridentifikasi, diperkirakan merupakan senyawa baru yaitu 3,5,4'-trimetoksi-6,7-metilendioksiflavon. Molecular docking antara casticin dan 2QMJ menghasilkan binding energy dan Ki terkecil -5,29 kkal/mol dan 131,54 mikromolar dengan interaksi ikatan hidrogen pada ARG202, ASN207, THR205, ASP542, dan TYR214 serta interaksi hidropobik LEU473, THR204, dan LEU473. Interaksi tricin dengan 3TOP menghasilkan binding energy dan Ki sebesar -8,02 kkal/mol dan 0,34 mikromolar melalui ikatan hidrogen dengan residu ASP1157, THR1586, HIS1584, dan ASP1279 serta interaksi hidropobik dengan TRP1355, TYR1251, PHE1559, TRP1418, TRP1523, HIS1584, ILE1315, dan ILE1280. Protein 3A4A berinteraksi ikatan hidrogen dengan centaureidin pada LYS373, ASN565, GLU562, dan LYS568 serta hidropobik dengan PRO567, LYS568, dan PHE494 menghasilkan binding energy dan Ki sebesar -8,02 kkal/mol dan 0,34 mikromolar. Casticin, tricin, dan centaureidin merupakan flavonoid dengan aktivitas terbaik dalam inhibisi alfa glukosidase.

A study of active compounds as a alpha glukosidase inhibitor from Buas-Buas leaves (Premna serratifolia) has been conducted. The research is based on the tradition of the West Borneo community of consuming P. serratifolia leaves as tonic, to treat headache, colic, diabetes, hypertension, difficulty breathing, malaria, and to increase breastmilk production. P. serratifolia extract is able to inhibit alpha glukosidase to decrease blood glucose levels. Because efforts in isolating secondary metabolites from P. serratifolia as alpha glukosidase inhibitors have not been conducted, a study is needed to determine the compound responsible for this activity which will be proven by in vitro and in silico. P. serratifolia leaves were dried, granulated, and macerated in ethanol (p.a) for 4 days. The obtained extract was fractioned in hexane, ethyl acetate, and water. All of the extracts were then determined of their alpha glukosidase inhibition activity, phytochemical, total phenolic (TPC), total flavonoid (TFC), and total steroid (TSC), and analysed with Fourier Transform Infrared (FTIR) spectroscopy. The effect of TPC, TFC, TSC, and functional groups on alpha glukosidase inhibition activity were determined by Principal Component Analysis (PCA) and Partial Least Square (PLS) regression. Isolation of active compounds from the fraction had the smallest IC50 using column chromatography and Preparative High Pressure Liquid Chromatography (preparative HPLC). The active compounds from the isolate were analysed by using Ultra High Performance Liquid Chromatography-Q Exactive hybrid quadrupole-Orbitrap High Resolution Mass Spectrometry (UHPLC-Q-Orbitrap HRMS). The interaction between alpha glukosidase and active compounds was studied by molecular docking utilizing N-terminal maltase glucoamylase (PDB code: 2QMJ), C-terminal maltase-glucoamylase (PDB code: 3TOP), and isomaltase (PDB code: 3A4A). The results of phytochemical screening show that there were flavonoids, phenolics, and saponins (steroids) in the ethanol extract, hexane, ethyl acetate, and water fraction. The results of in vitro test show that ethyl acetate fraction has the best inhibition with IC50 10900 ppm. Based on PCA and PLS analyses, the highest standardization coefficient was shown by TFC and -C=O functional groups therefore they have the biggest contribution to inhibit alpha glukosidase. F12-F14 subfractions from ethyl acetate fraction were performed the lowest IC50 (< 2000 ppm). The analysis of UHPLC-Q-Orbitrap HRMS show that nine flavonoids were detected which are centaureidin, chyrsin, pectolinaringenin, glycitein, kaempferide, syringetin, tricin, casticin, and 3,5,4'-trimethoxy-6,7-methylenedioxyflavone. One of the 9 flavonoids that had been identified, is estimated to be a new compound, namely 3,5,4'-trimethoxy-6,7-methylenedioxyflavone. Casticin was found to interact with 2QMJ by ARG202, ASN207, THR205, ASP542, and TYR214 via hydrogen bonding and hydrophobic interactions with LEU473, THR204, and LEU473, binding energy and Ki of -5.29 kcal/mol and 131.54 micromolar. Meanwhile, tricin showed binding energy and Ki -6.77 kcal/mol and 10.89 micromolar to 3TOP for the hydrogen bonding interaction with ASP1157, THR1586, HIS1584, and ASP1279, and hydrophobic interactions involving TRP1355, TYR1251, PHE1559, TRP1418, TRP1523, HIS1584, ILE1315, and ILE1280. Centaureidin showed the lowest binding energy and Ki of -8.02 kcal/mol and 0.34 micromolar with LYS373, ASN565, GLU562, and LYS568 interacting with hydroxyl and carbonyl groups via hydrogen bonding; PRO567, LYS568, and PHE494 residues interacted via hydrophobic interaction. Casticin, tricin, and centaureidin are the best flavonoids that inhibit alpha glukosidase.

Kata Kunci : Premna serratifolia, alpha glukosidase, flavonoid, molecular docking