Biflavonoid dari Araucaria cunninghamii Mudie Indonesia dan Potensinya Sebagai Agen Sitotoksik Penghambat Proteasom

Abstract

LUTHFAN IRFANA. Biflavonoid dari Araucaria cunninghamii Mudie Indonesia dan Potensinya sebagai Agen Sitotoksik Penghambat Proteasom. Dibimbing oleh PURWANTINIGSIH SUGITA, SETYANTO TRI WAHYUDI, SUMINAR SETIATI ACHMADI, dan BUDI ARIFIN. Araucaria adalah genus pohon konifera yang berasal dari belahan bumi selatan. Pohon genus Araucaria telah dimanfaatkan sebagai sumber kayu dan, secara lebih terbatas, sebagai bahan nutrisi serta obat tradisional. Beberapa spesiesnya berstatus terancam punah. Di kawasan barat Indonesia, Kebun Raya Bogor dan Taman Bunga Nusantara memiliki koleksi berbagai spesies pohon Araucaria. Perbedaan iklim dan kondisi lokasi tumbuh dari habitat aslinya menyiratkan kemungkinan perbedaan kandungan metabolit sekunder yang belum tereksplorasi. Sebelumnya, kelompok penelitian kami telah mengisolasi tujuh biflavonoid dari daun kering Araucaria hunsteinii K. Schuum dari lokasi-lokasi tersebut. Salah satu biflavonoid yang terisolasi, yaitu isoginkgetin, menunjukkan aktivitas sitotoksik yang menjanjikan pada galur sel kanker payudara MCF7. Biflavonoid adalah kelas senyawa bahan alam kaya aktivitas hayati yang umumnya berupa dimer dari unit-unit flavonoid seperti apigenin. Sementara itu, spesies A. cunninghamii Mudie (Damar laki-laki) dari koleksi tersebut belum pernah dieksplorasi potensi fitokimianya. Target biomolekul utama dari sebagian besar biflavonoid tersebut juga belum diketahui. Hanya isoginkgetin yang pernah dilaporkan memicu apoptosis sel kanker melalui penghambatan aktivitas proteolitik subunit ß1, ß2, dan ß5 dari proteasom. Partikel inti dari proteasom (20S) mengandung beberapa aktivitas proteolitik yang berhubungan erat dengan kelangsungan hidup sel. Dalam sel kanker, proteasom jauh lebih aktif sehingga penghambatannya lebih rentan memicu kematian sel. Tujuan penelitian ini adalah melengkapi data struktur dan aktivitas biflavonoid genus Araucaria Indonesia, khususnya dari daun spesies A. cunninghamii yang tumbuh di Taman Bunga Nusantara, Indonesia. Aktivitas yang diukur adalah sitotoksisitas (in vitro) terhadap sel kanker payudara MCF7 dan konsentrasi enzim proteasomnya. Penelitian ini juga bertujuan memprediksi potensi aktivitas penghambatan proteasom oleh biflavonoid A. cunninghamii melalui penambatan molekul, hubungan kuantitatif struktur-aktivitas (QSAR), serta dinamika molekul (MD). Sampel daun kering A. cunninghamii Mudie diekstraksi dengan aseton pada suhu kamar, diambil fraksi polarnya, lalu dipisahkan dari tanin. Fraksi polar dimurnikan lebih lanjut dengan kromatografi kolom vakum dengan fase diam Sephadex dan fase gerak metanol. Fraksi flavonoid selanjutnya dimurnikan dengan kromatografi radial elusi gradien dengan fase diam gel silika. Struktur senyawa terisolasi ditentukan melalui berbagai pengukuran spektroskopi. Aktivitas in-vitro senyawa terisolasi diukur melalui pengujian toksisitas terhadap galur sel MCF7 menggunakan pewarna 3-(4,5-dimetiltiazol-2-il)-2,5-difeniltetrazolium bromida (MTT). Konsentrasi proteasom contoh sel lalu diukur dengan kit uji ELISA (spesifik subunit a6). Untuk menjelaskan tren yang diperoleh, berbagai model struktur molekul biflavonoid terisolasi ditambatkan secara in silico pada struktur target PDB 3MG8 (proteasom ragi dengan bagian anulus a terbuka) dan 5LE5 (bagian kantung K subunit a6 proteasom manusia) dengan AutoDock-GPU. Hubungan antara nilai penambatan dan uji ELISA lalu dimodelkan secara linear. Tiga biflavonoid yang telah diketahui berhasil diisolasi dan ditentukan struktur molekulnya dalam penelitian ini. Senyawaan tersebut adalah 7,4',7?,4?-tetra-O-metilkupresuflavon (1), 7,4',4?-tri-O-metilrobustaflavon (2), dan 7,7??-di-O-metilamentoflavon (3). Di antara biflavonoid yang diisolasi, senyawa 3 menunjukkan estimasi aktivitas in vitro terbaik (IC50 = 150,04 ± 23,97 µM) diikuti oleh 1 (IC50 > 1000 µM), dan 2 (IC50 > 2000 µM). Dengan demikian, aktivitas ketiga isolat dikategorikan lemah (IC50 > 10 µM). Semua biflavonoid yang terisolasi juga ditemukan berdampak lemah pada konsentrasi proteasom MCF7. Senyawa 3 menurunkan 32% konsentrasi proteasom MCF7, relatif terhadap kontrol negatif, pada konsentrasi perlakuan 25 µg/mL. Di lain pihak, 1 dan 2 berturut-turut mengurangi 28 dan 16% konsentrasi proteasom pada konsentrasi perlakuan 1000 µg/mL. Telaah in silico menunjukkan nilai penambatan molekul 1–3 pada reseptor kantung sisteina di antara subunit a6 dan a3 PDB 5LE5 berpengaruh signifikan pada tren peringkat respons uji ELISA (konsentrasi signifikan 3 < 1 < 2). Hasil ini menyarankan bahwa uji ELISA yang dilakukan belum dapat menggambarkan potensi penghambatan aktivitas proteolitik. Potensi biflavonoid A. cunninghamii (1–3) sebagai inhibitor proteolitik non-kovalen subunit ß5 proteasom 20S diprediksi dengan penambatan model struktur molekul biflavonoid terisolasi pada target PDB 4RUR. Reseptor 4RUR adalah struktur kristal proteasom 20S ragi yang terkompleks dengan inhibitor indolo-fakelin (3WE) pada bagian subunit ß5-nya. Selanjutnya, pose terbaik dari penambatan disimulasikan dengan dinamika molekul (MD) selama 100 ns. Terakhir, pemodelan kuadrat terkecil parsial (PLS) berbasis hubungan struktur dan aktivitas kuantitatif tiga dimensi (QSAR 3D) dilakukan untuk menduga IC50 biflavonoid terisolasi. Model PLS dibuat menggunakan data latih berupa model struktur 3D dan IC50 senyawa-senyawa yang telah dilaporkan aktivitas penghambatan proteasomnya (subunit ß5). Penambatan pada situs pengikatan 3WE di subunit ß5 menapis bentuk tautomer dari 1–3 dengan nilai penambatan terbaik. Senyawa 2 dan 3 terletak lebih dekat ke residu treonina ujung-N (Thr1) yang vital untuk fungsi proteolitik daripada 3WE dan isoginkgetin. Setelah simulasi MD, hanya kedekatan antara gugus 4'-OH dari 3 dan Thr1 yang diprediksi bertahan selama 14% waktu simulasi, menyiratkan interaksi yang reversibel. Luaran dari penelitian ini telah diterbitkan di berkala ilmiah Trends in Sciences vol. 22 no. 3 dan Tropical Journal of Natural Product Research vol. 9 no. 5. Kebaruan yang dicapai penelitian ini adalah isolasi 7,4',4?-tri-O-metilrobustaflavon dan 7,7?-di-O-metilamentoflavon dari daun kering spesies A. cunninghamii Mudie. Kajian in silico dalam penelitian ini masih sangat terbatas dan memerlukan validasi eksperimental untuk menelaah akurasinya. Namun demikian, kerangka senyawa amentoflavon dari A. cunninghamii diramalkan lebih potensial untuk dikembangkan sebagai perancah inhibitor non-kovalen proteasom daripada kerangka kupresuflavon dan robustaflavon dalam penelitian ini.

LUTHFAN IRFANA. Biflavonoids from Araucaria cunninghamii Mudie Indonesia and Their Potential as Cytotoxic Agent Proteasome Inhibitors. Supervised by PURWANTINIGSIH SUGITA, SETYANTO TRI WAHYUDI, SUMINAR SETIATI ACHMADI, and BUDI ARIFIN. Araucaria is a genus of coniferous trees native to the southern hemisphere. Trees of the genus Araucaria have been exploited as a source of timber and, to a lesser extent, as a source of nutrition and traditional medicine. Several species of the genus are endangered. In western Indonesia, the Kebun Raya Bogor and the Taman Bunga Nusantara have collections of various species of Araucaria trees. The differences in climate and growing conditions than the native habitat imply the possibility of variation in secondary metabolite content that have not been explored. Previously, our research group has isolated seven biflavonoids from dried leaves of Araucaria hunsteinii K. Schuum from these locations. One of the isolated biflavonoids, isoginkgetin, showed promising cytotoxic activity on the breast cancer cell line MCF7. Biflavonoids are a class of natural compounds rich in biological activity that are generally dimers of flavonoid units such as apigenin. Meanwhile, the species A. cunninghamii Mudie (Hoop Pine) of the collection has never been explored for its phytochemical potential. The main biomolecular targets of most of these biflavonoids are also unknown. Only isoginkgetin has been reported to trigger apoptosis of cancer cells through inhibition of the proteolytic activity of the ß1, ß2, and ß5 subunits of the proteasome. The core particle of the proteasome (20S) contains several proteolytic activities that are closely related to cell survival. In cancer cells, the proteasome is much more active so that its inhibition is more likely to trigger cell death. The purpose of this study was to enrich the data on the structure and activity of biflavonoids of the genus Araucaria Indonesia, especially from the leaves of the A. cunninghamii species growing in Taman Bunga Nusantara, Indonesia. The activities measured were cytotoxicity (in vitro) against MCF7 breast cancer cells as well as the concentration of the proteasome enzymes. This study also aims to predict the potential for proteasome inhibitory activity by A. cunninghamii biflavonoids through molecular docking, quantitative structure-activity relationships (QSAR), and molecular dynamics (MD). Dried leaf samples of A. cunninghamii Mudie were extracted with acetone at room temperature, the polar fraction was taken, and then separated from the tannins. The polar fraction was further purified by vacuum column chromatography with Sephadex stationary phase and methanol mobile phase. The flavonoid fraction was further purified by radial gradient elution chromatography with silica gel stationary phase. The structure of the isolated compounds was determined through various spectroscopic measurements. The in-vitro activity of the isolated compounds was measured through toxicity testing against MCF7 cell lines using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) dye. The proteasome concentration of the cell samples was then measured using an ELISA test kit (specific subunit a6). To explain the obtained trends, various molecular structure models of isolated biflavonoids were docked in silico to the PDB target structures 3MG8 (yeast proteasome with open a-annulus region) and 5LE5 (K-pocket region of human proteasome a6 subunit) using AutoDock-GPU. The relationship between docking values and ELISA assay was then linearly modeled. Three known biflavonoids were successfully isolated and their molecular structures determined in this study. These compounds are 7,4',7?,4?-tetra-O-methylcupressuflavone (1), 7,4',4?-tri-O-methylrobustaflavone (2), and 7,7?-di-O-methylamentoflavone (3). Among the isolated biflavonoids, compound 3 showed best in vitro activity estimate (IC 50 = 150.04 ± 23.97 µM) followed by 1 (IC50 > 1000 µM), and 2 (IC50 > 2000 µM). Thus, the activity of the three isolates was categorized as weak (IC 50 > 10 µM). All isolated biflavonoids were also found to have a weak effect on the concentration of MCF7 proteasomes. Compound 3 decreased 32% of the concentration of MCF7 proteasomes, relative to the negative control, at a treatment concentration of 25 µg/mL. On the other hand, 1 and 2 decreased 28 and 16% of the concentration of proteasomes, respectively, at a treatment concentration of 1000 µg/mL. In silico study showed that the molecular docking values 1–3 on the cysteine pocket around the PDB 5LE5 a6 subunit had a significant effect on the ELISA response (significant concentration 3 < 1 < 2). These results suggest that the ELISA test performed cannot yet describe the potential inhibition of proteolytic activity of the proteasome by 1–3. A. cunninghamii biflavonoids (1–3) as non-covalent proteolytic inhibitors of the ß5 subunit of the 20S proteasome was predicted by docking the molecular structure model of the isolated biflavonoids to the PDB target 4RUR. The 4RUR receptor is a crystal structure of the yeast 20S proteasome complexed with indolo-phakelline (3WE) on its ß5 subunit. Furthermore, the best pose of the docking was simulated by molecular dynamics (MD) for 100 ns. Finally, partial least squares (PLS) modeling based on three-dimensional quantitative structure-activity relationship (3D QSAR) was performed to predict the IC50 of isolated biflavonoids. The PLS model was created using training data in the form of 3D structure models and IC50 compounds that have been reported for their proteasome inhibitory activity (ß5 subunit). Docking at the 3WE binding site in the ß5 subunit screened tautomeric forms of 1–3 with the best docking values. Compounds 2 and 3 are located closer to the N-terminal threonine residue (Thr1) which is vital for proteolytic function than 3WE and isoginkgetin. After MD simulations, only the proximity between the 4?-OH group of 3 and Thr1 is predicted to persist for 14% of the simulation time, implying a reversible interaction. The output of this research has been published in the scientific periodical Trends in Sciences. vol. 22 no. 3 and Tropical Journal of Natural Product Research vol. 9 no. 5. The novelty achieved in this research is the isolation 7,4',4?-tri-O-methylrobustaflavone and 7,7?-di-O-methylamentoflavone from the dried leaves of the species A. cunninghamii Mudie. The in-silico study in this study is still very limited and requires experimental validation to examine its accuracy. However, the amentoflavone compound framework from A. cunninghamii is predicted to be more potential to be developed as a non-covalent proteasome inhibitor scaffold than the cupressuflavone and robustaflavone frameworks in this study.