Farahdina, Afifah Aulia and Prof. Dr. dr. Loeki Enggar Fitri, M.Kes, Sp.ParK and Prof. Agustina Tri Endharti, Ssi., Ph.D. (2024) Profiling Senyawa Aktif Fraksi 42 Ekstrak Metabolit Sekunder Bakteri Streptomyces Hygroscopicus Subsp. Hygroscopicus Sebagai Antimalaria Menggunakan Liquid Chromatography Mass Spectrometry (Lcms) Dan Molecular Docking (In Silico). Magister thesis, Universitas Brawijaya.
Abstract
Malaria merupakan penyakit menular yang ditularkan melalui nyamuk Anopheles betina yang terinfeksi Plasmodium dan dapat menyebabkan komplikasi yang mengancam jiwa. Resistensi Plasmodium falciparum terhadap obat antimalaria merupakan masalah nyata yang dihadapi dunia. Salah satu penyebab tingginya angka kesakitan dan kematian adalah meningkatnya angka resistensi ini sehungga pengembangan obat antimalaria baru sangat diperlukan untuk mengatasi resistensi antimalaria yang semakin meningkat tersebut. Streptomyces adalah genus bakteri gram positif di tanah yang banyak dipelajari diantara mikroorganisme lain. Streptomyces mampu menghasilkan banyak bioaktif metabolit sekunder yang bermanfaat bagi manusia. Penelitian membuktikan bahwa ekstrak metabolit sekunder Streptomyces hygroscopicus Subsp. Hygroscopicus memiliki efek antimalaria secara in vivo dan in silico. Beberapa fraksi dari hasil fraksinasi pertama ekstrak metabolit sekunder Streptomyces hygroscopicus Subsp. Hygroscopicus pada penelitian sebelumnya menunjukkan penghambatan enzim mitokondria Plasmodium falciparum Malate Quinone Oxidoreductase (PfMQO) secara in vitro. Proses fraksinasi ekstrak metabolit Streptomyces hygroscopicus Subsp. Hygroscopicus pada penelitian ini merupakan fraksinasi replikasi kedua yang dilakukan dengan prosedur yang sama dengan fraksinasi pertama. Fraksinasi dilakukan dengan alat BUCHI Reveleris® PREP Purification System yang menghasilkan 60 fraksi. Pemilihan fraksi 42 ekstrak metabolit Streptomyces hygroscopicus Subsp. Hygroscopicus pada penelitian ini didasarkan pada hasil penelitian in vivo sebelumnya. Penelitian sebelumnya menyatakan bahwa fraksi 42 Strepyomyces hygroscopicus dapat menekan derajat parasitemia, menaikkan jumlah persentase inhibisi pertumbuhan parasit secara dose dependent. Penelitian in silico yang menunjukkan fraksi 44 ekstrak metabolit Streptomyces hygroscopicus Subsp. Hygroscopicus mengandung senyawa Trypthantrin yang memilki nilai afinitas ikatan lebih tinggi dengan protein Plasmodium falciparum yang bekerja di food vacuole. Penelitian ini bertujuan untuk mengidentifikasi senyawa yang terkandung dalam fraksi 42 ekstrak metabolit Streptomyces hygroscopicus Subsp. Hygroscopicus menggunakan Liquid Chromatography Mass Spectrometry (LCMS) dan menganalisis aktivitas antimalarial fraksi 42 ekstrak metabolit Streptomyces hygroscopicus Subsp. Hygroscopicus secara in silico. Hasil analisis kandungan senyawa dalam fraksi 42 menjadi dasar analisis in silico yang mentarget senyawa aktif kepada protein spesifik Plasmodium falciparum yang bekerja di food vacuole yaitu Plasmodium falciparum Aminopeptidase-M1 (PfA-M1), Plasmodium falciparum Chloroquine Resistance Transporter (PfCRT), Falcipain-2 Protease.x Dari hasil LCMS fraksi 42 ekstrak metabolit Streptomyces hygroscopicus Subsp. Hygroscopicus dari empat puncak tertinggi dianalisis dengan retention time 0–20 menit, ada empat senyawa yang dapat diidentifikasi yaitu Dibenzylamine, Sedanolide, Levalbuterol, dan Dibutyl phthalate. Analisis in silico dilakukan dengan metode molecular docking menggunakan PfA-M1, PfCRT, dan Falcipain-2 Protease sebagai target protein. Preparasi target protein dilakukan dengan mengunduh protein dari database Protein Data Bank (PDB) dan senyawa aktif ligan dari PubChem. Dari analisis farmakokinetik menggunakan SwissADME terbukti bahwa keempat senyawa tersebut memenuhi kriteria kemiripan obat berdasarkan Lipinski’s rule of five dan dan Veber’s Rule. Hasil penelitian in silico menunjukkan bahwa senyawa Dibenzylamine, Sedanolide, Levalbuterol, Dibutyl phthalate memiliki nilai afinitas ikatan dengan protein PfA-M1 secara berturut-turut sebesar -7,4 Kkal/mol, -6,9 Kkal/mol, -6,9 Kkal/mol, -6,8 Kkal/mol, ikatan keempat senyawa tersebut dengan PfCRT berturut-turut sebesar -6,7 Kkal/mol, -6,4 Kkal/mol, -6,5 Kkal/mol, -6,4 Kkal/mol, ikatan keempat senyawa tersebut dengan Falcipain-2 Protease berturut-turut sebesar -6,4 Kkal/mol, -5,5 Kkal/mol, -5,8 Kkal/mol, -4,9 Kkal/mol. Nilai afinitas ikatan yang dihasilkan mirip dengan ikatan target protein PfA-M1, PfCRT, Falcipain-2 Protease dan ligan kontrol yang secara berturut-turut sebesar -8,1 Kkal/mol, -7,3 Kkal/mol, dan -7,1 Kkal/mol. Hasil interaksi ikatan senyawa fraksi 42 ekstrak metabolit Streptomyces hygroscopicus Subsp. Hygroscopicus memiliki ikatan hidrogen dan hidrofobik dengan asam amino yang sama pada ikatan protein target dan ligan kontrol. Dibenzyalmine memiliki nilai rata-rata RMSD yang hampir sama dengan kontrol yaitu 1,28 Å (Dibenzylamine-PfA-M1) dan 1,27 Å (Kontrol-PfA-M1), RMSF dengan residu asam amino yang berperan paling banyak dibandingkan dengan kontrol, dan nilai binding energy lebih kecil dari kontrol yaitu 523 Kkal/mol (PfA-M1- Dibenzylamine) dan 675 Kkal/mol (PfA-M1-kontrol). Dari penelitian ini dapat disimpulkan bahwa fraksi 42 ekstrak metabolit Streptomyces hygroscopicus Subsp. Hygroscopicus yang mengandung Dibenzylamine, Sedanolide, Levalbuterol, Dibutyl phthalate cukup berpotensi sebagai agen antimalaria. Selain itu, dengan karakteristik ikatan yang cukup kuat pada transporter PfA-M1, PfCRT, Falcipain-2 Protease, keempat senyawa tersebut memiliki ikatan hidrogen dan ikatan hidrofobik pada asam amino yang sama dengan protein target dan ligan kontrol. Senyawa Dibenzylamine merupakan senyawa dengan nilai afinitas ikatan tertinggi (-7,4 Kkal/mol) dibandingkan senyawa lain pada seluruh protein target dan merupakan struktur yang lebih stabil dan fleksibel dibandingkan kontrol (rata-rata RMSD 1,28 Å dan binding energy 523 Kkal/mol). Dibutuhkan penelitian lebih lanjut untuk mengembangkan penelitian kandidat antimalarial dari fraksi 42 ekstrak metabolit Streptomyces hygroscopicus Subsp. Hygroscopicu
English Abstract
Malaria is an infectious disease that attacks humans and animals which is transmitted through female Anopheles mosquitoes infected with Plasmodium and cause life-threatening complications. Plasmodium falciparum resistance to antimalarial drugs is a real problem facing the world. One of the causes of high morbidity and mortality rates is increasing resistance rates. The development of new antimalarial drugs is urgently needed to overcome the increasing antimalarial resistance. Streptomyces is a genus of gram-positive bacteria in soil that is widely studied among other microorganisms. Streptomyces is capable of producing many new bioactive secondary metabolites that are beneficial to humans. Preliminary research was found that the secondary metabolite extract of Streptomyces hygroscopicus Subsp. Hygroscopicus has been proven to have antimalarial effects in vivo and in silico. Several fractions from the results of the first fractionation of the secondary metabolite extract of Streptomyces hygroscopicus Subsp. Hygroscopicus in previous research showed inhibition of the mitochondrial enzyme Plasmodium falciparum Malate Quinone Oxidoreductase (PfMQO) in vitro. Fractionation process of metabolite extract of Streptomyces hygroscopicus Subsp. Hygroscopicus is the second replication fractionation carried out using the same procedure as the first fractionation. Fractionation was carried out using a BUCHI Reveleris® PREP Purification System which produced 60 fractions. Fraction selection of 42 metabolite extracts of Streptomyces hygroscopicus Subsp. Hygroscopicus in this study is based on the results of previous in vivo research. Previous research states that fraction 42 of Strepyomyces hygroscopicus can reduce the degree of parasitemia dan increase the percentage of parasite growth inhibition in a dose dependent manner. Previous in silico research showed that fractions of 44 metabolite extracts of Streptomyces hygroscopicus Subsp. Hygroscopicus contains the Trypthantrin compound which has a higher binding affinity value with the Plasmodium falciparum protein which works in the food vacuole. This study aimeds to identify the compounds contained in the 42 metabolite extract fractions of Streptomyces hygroscopicus Subsp. Hygroscopicus used Liquid Chromatography Mass Spectrometry (LCMS) and analyzed the antimalarial activity of 42 metabolite extracts from Streptomyces hygroscopicus Subsp. Hygroscopicus in silico. The results of the analysis of compound content in fraction 42 became the basis for in silico analysis which targets active compounds to specific Plasmodium falciparum proteins that work in the food vacuole, namely Plasmodium falciparum Aminopeptidase-M1 (PfA-M1), Plasmodium falciparum Chloroquine Resistance Transporter (PfCRT), Falcipain-2 Protease. From the LCMS results of fraction 42 metabolite extracts of Streptomyces hygroscopicus Subsp. Hygroscopicus from the four highest peaks analyzed withxii retention time 0–20 minutes, there were four compounds that could be identified, namely Dibenzylamine, Sedanolide, Levalbuterol, and Dibutyl phthalate. In silico analysis was carried out using the molecular docking method using PfA-M1, PfCRT, and Falcipain-2 Protease as protein targets. Protein target preparation was carried out by downloading proteins from the Protein Data Bank (PDB) database and ligand active compounds from PubChem. From pharmacokinetic analysis using SwissADME, it was proven that the four compounds met the drug similarity criteria based on Lipinski's rule of five and Veber's Rule. The results of in silico research show that the compounds Dibenzylamine, Sedanolide, Levalbuterol, Dibutyl phthalate have binding affinity values with the PfA-M1 protein of -7.4 Kcal/mol, -6.9 Kcal/mol, -6.9 Kcal, -6.8 Kcal/mol respectively, the bonds of the four compounds with PfCRT are respectively -6.7 Kcal/mol, -6.4 Kcal/mol, -6.5 Kcal/mol, -6.4 Kcal /mol, the binding of these four compounds with Falcipain-2 Protease is -6.4 Kcal/mol, -5.5 Kcal/mol, -5.8 Kcal/mol, -4.9 Kcal/mol, respectively. The resulting binding affinity values are similar to the binding protein targets PfAM1, PfCRT, Falcipain-2 Protease and control ligands which are respectively -8.1 Kcal/mol, -7.3 Kcal/mol, and -7.1 Kcal/mol. The results of the compound bond interactions of fraction 42 metabolite extracts of Streptomyces hygroscopicus Subsp. Hygroscopicus has hydrogen and hydrophobic bonds with the same amino acids in target protein binding and control ligands. Dibenzyalmine has an average RMSD value that is almost the same as the control, 1.28 Å (Dibenzylamine-PfAM1) and 1.27 Å (Control-PfA-M1), RMSF with amino acid residues that play the most role compared to the control, and the binding energy value is smaller than the control, namely 523 Kcal/mol (PfA-M1- Dibenzylamine) and 675 Kcal/mol (PfAM1-control). From this research it can be concluded that fraction 42 of the metabolite extract of Streptomyces hygroscopicus Subsp. Hygroscopicus which contains Dibenzylamine, Sedanolide, Levalbuterol, Dibutyl phthalate has potential as an antimalarial agent. In addition, with fairly strong binding characteristics in the PfAM1, PfCRT, Falcipain-2 Protease transporter, these four compounds have hydrogen bonds and hydrophobic bonds in the same amino acids as the target protein and control ligand. The Dibenzylamine compound is the compound with the highest binding affinity value (-7.4 Kcal/mol) compared to other compounds in all target proteins and is a more stable and flexible structure than the control (average RMSD 1.28 Å and binding energy 523 Kcal/ moles).Further research is needed to develop antimalarial candidate research from the 42 metabolite extract fractions of Streptomyces hygroscopicus Subsp. Hygroscopicus.
| Item Type: | Thesis (Magister) |
|---|---|
| Identification Number: | 0424060099 |
| Divisions: | S2/S3 > Magister Ilmu Biomedis, Fakultas Kedokteran |
| Depositing User: | Unnamed user with username nova |
| Date Deposited: | 09 Oct 2024 06:23 |
| Last Modified: | 09 Oct 2024 06:23 |
| URI: | http://repository.ub.ac.id/id/eprint/231505 |
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