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Abstract
The fibrinolytic protease enzyme is an enzyme that has the ability to break down blood clots that can cause cardiovascular disease. This enzyme can be found in pucuk merah (Syzygium myrtifolium Walp), which is a popular plant and easy to find. The aim was to determine the total protein content, protein molecular weight, and fibrinolytic activity of pucuk merah leaf enzyme extract which were tested using fibrin plate and clot lysis methods. The stages of the research included enzyme extraction using 0.1 M phosphate buffer pH 7, precipitation of crude enzyme extract with 80% ammonium sulfate, dialysis of enzyme extract using 12-14 kDa cut-off dialysis bags, enzyme extract characterization using SDS-PAGE, total protein measurement using the Bradford method and testing the fibrinolytic activity of enzyme extracts using the fibrin plate and clot lysis methods. Positive and negative controls are nattokinase and phosphate buffer. The fibrinolytic activity of the enzyme extract was analyzed by one-way analysis of variance (ANOVA) using SPSS 26 software. The highest total protein content was in the enzyme extract precipitate sample of 1864 μg/ml. The molecular weight of the fibrinolytic enzyme extract of pucuk merah leaves ranges from 25 to 45 kDa. The precipitate sample of pucuk merah leaf enzyme extract had the highest fibrinolytic activity against fibrin plate testing of 11.23 mm and clot lysis testing of 13.90%. Enzyme extract precipitate is the most effective sample because it has the highest fibrinolytic activity.
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Keywords: clot lysis, fibrinolytic, fibrin plate, pucuk merah
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References
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- Rahmi, Hanifah., Hariyanti., A, Rina Putri., Wulandari, Devi. (2020). Analisis Hasil Fraksinasi Protease dan Lipase yang Berasal dari Saluran Pencernaan Udang Vaname (Litopenaeus vannamei). Jurnal Bioteknologi & Biosains Indonesia, 7(2), pp. 194–202.
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References
Abedin, Fokhrul., Hussain, Md. Saddam., Islam, Areful., Sen, Niloy., Das, Abhijit., Kar, Auditi., Hossain, Md. Monir., Millat, Md. Shalahuddin. & Islam, Mohammad Safiqul. (2018). Thrombolytic, CNS depressant and anti-diarrhoeal activities of ethanolic extract of bark of Syzygium cumini L. skeels: An in-vivo and in-vitro study. Journal of Pharmacy and Nutrition Sciences. Lifescience Global, 8(3), pp. 129–136. doi: 10.6000/1927-5951.2018.08.03.7.
Antao, C. M. & Malcata, F. X. (2005). Plant serine proteases: Biochemical, physiological and molecular features. Plant Physiology and Biochemistry. Elsevier Masson SAS, pp. 637–650. doi: 10.1016/j.plaphy.2005.05.001.
Chadni, S. H., Hasan, A. Al & Azam, A. Z. (2015). Antimicrobial, Cytotoxic, Thrombolytic and Antioxidant Activities of Syzygium fruticosum (Roxb.) DC. Bangladesh Pharmaceutical Journal. Bangladesh Journals Online (JOL), 17(1), pp. 51–54. doi: 10.3329/bpj.v17i1.22315.
Chen, Hongjie., McGowan, Eileen M., Ren, Nina., Lal, Sara., Nassif, Najah., Kaneez, Fatima Shad., Qu, Xianqin. & Lin, Yiguang. (2018). Nattokinase: A Promising Alternative in Prevention and Treatment of Cardiovascular Diseases. Biomarker Insights. SAGE Publications Ltd. doi: 10.1177/1177271918785130.
Chung, Dong-Min., Nack-Shick, Choi., Hyo, Kon Chun., Pil, Jae Maeng., Sang-Bong, Park., & Sung-Ho, Kim. (2010). A New Fibrinolytic Enzyme (55 kDa) from Allium tuberosum: Purification, Characterization, and Comparison. Journal of Medicinal Food, 13(6), pp. 1532–1536
Escobar., Harmaening., Joiner., Simmons., Smith., & Moore. (2002). Introduction to Hemostasis. In Hermaening D.M (ed.) Clinical Hematology and Fundamentals of Hemostasis. 4th edn. FA Davis, Philadelphia.
Garfin, DE., Davey, J., & Lord, M. (2003). Essential Cell Biology: Cell Structure, A Practical Approach. United Kingdom: Oxford University Press.
Harahap, N. S. (2014). Protein dalam Nutrisi Olahraga. Jurnal Ilmu Keolahragaan, 13(2), pp. 45-54
Institute for Health Metrics and Evaluation. 2019. Cardiovascular Disease Statistics in Indonesia. Institute for Health Metrics and Evaluation (IHME).
Kementrian Kesehatan RI. (2019). Laporan Nasional RISKESDAS 2018. Jakarta: Kementrian Kesehatan Republik Indonesia: Badan Penelitian dan Pengembangan Kesehatan.
Khopkar. (2007). Konsep Dasar Kimia Analitik. Jakarta: UI-Press.
Kim, Dae-Won., Jun-Hui, Choi., Se-Eun, Park., Seng, Kim., Kumar, Sapkota. & Sung-Jun, Kim. (2015). Purification and characterization of a fibrinolytic enzyme from Petasites japonicus. International Journal of Biological Macromolecules. Elsevier B.V., 72, pp. 1159–1167. doi: 10.1016/j.ijbiomac.2014.09.046.
Kotb, Essam. (2013). Activity assessment of microbial fibrinolytic enzymes. Applied Microbiology and Biotechnology, pp. 6647–6665. doi: 10.1007/s00253-013-5052-1.
Kumaunang, M., & Kamu, V. (2011). Aktivitas Enzim Bromelin dari Ekstrak Kulit Nanas (Anenas comosus). Jurnal Ilmiah Sains, 11(2), pp. 198-201
Kusumo, A.R., Poernomo, A.T. & Primaharinastiti, R. (2021). Aktivitas Agen Trombolitik Daun Pegagan (Centella asiatica) Hasil Fermentasi oleh Acetobacter aceti FNCC 0016 dengan Metode Clot Lysis secara In-Vitro. Jurnal Ilmiah Manuntung, 7(2), pp. 215–223.
Masyitoh, M. D., Dewanti, I.D.A.R. & Setyorini, D. (2016). Analisis Profil Protein Ekstrak Aquades dan Etanol Daun Mimba (Azadirachta Indica A. Juss) dengan Metode SDS-PAGE (Protein Profile Analysis of Aquadest and Ethanol Extract of Neem Leaves by Means of SDS-PAGE Method), Jurnal Pustaka Kesehatan, 4(3), pp. 533-540
Matsubara, Kiminori., Kanji, Hori., Yasushi, Matsuura., Keisuke, Miyazawa. (2000). Purification and characterization of a fibrinolytic enzyme and identification of fibrinogen clotting enzyme in a marine green alga, Codium divaricatum. Comparative Biochemistry and Physiology Part B, 125, pp. 137-143. Available at: www.elsevier.com/locate/cbpb.
Mazid, A., & Al-Mamun, M. R. (2012). Thrombolytic activity of some spices and plants available in Bangladesh Synthesis of Antibiotic-Nanoparticle conjugates and Its Functional Characterization against Resistant Bacteria View project Phytopharmacological Screening of Bangladeshi herbs. Article in Thai Journal of Pharmaceutical Sciences, 36, pp. 72-77. Available at: https://www.researchgate.net/publication/260341390.
Nuraini, L., Purwanto, B.T., Syahrani, A., Primaharinastiti, R., & Poernomo, A.T. (2021). Pengaruh Proses Fermentasi pada Daun Centella asiatica oleh Acetobacter tropicalis terhadap Aktivitas Trombolitik. Majalah Farmasetika 6 (Suppl 1), pp. 1–13.
Patel, Divyang., Sapna, Desai., Ankita, Desai, Devanshi, Dave. & Dhananjay, Meshram. (2019). Phytochemical evaluation and In-vitro thrombolytic activity of hydro alcoholic extract of Syzygium malaccense leaves. Journal of Pharmacognosy and Phytochemistry, 8(3), pp. 3916-3918
Poernomo, Achmad Toto., Isnaeni. & Purwanto. (2015). Aktivitas in-vitro enzim fibrinolitik ekstrak tempe hasil fermentasi Rhizopus oligosporus ATCC 6010 pada substrat kedelai hitam. Berkala Ilmiah Kimia Farmasi, 4(2), pp. 18-24.
Rahmi, Hanifah., Hariyanti., A, Rina Putri., Wulandari, Devi. (2020). Analisis Hasil Fraksinasi Protease dan Lipase yang Berasal dari Saluran Pencernaan Udang Vaname (Litopenaeus vannamei). Jurnal Bioteknologi & Biosains Indonesia, 7(2), pp. 194–202.
Sajuthi, Dondin., Irrma, Suparto., Yanti., & Willy, Praira. (2010). Purifikasi dan Pencirian Enzim Protease Fibrinolitik dari Ekstrak Jamur Merang. Makara Sains, 14(2), pp. 145-150. Availableat:https://scholarhub.ui.ac.id/science/vol14/iss2/7.
Suranto. (2006). Electrophoresis Studies of Ranunculus Triplodantus Population. Biodiversitas, 1(1), pp. 1–7.
Tarek Md. Ibrahim., Hasanat, Abul., Kabir, Mohammad Shah Hafez., Chowdhury, Tanvir Ahmad., Rahman, Md. Mizanur. & Hossain, Dr. Mir Ezharul. (2015). Title In-vitro thrombolytic and cytotoxic activity of methanolic extract of Syzygium operculatum leaves. The Journal of Phytopharmacology, 4(2), pp. 87–89.
Thomas, B. R., van Deynze, A. & Bradford, K. J. (2002). Production of Therapeutic Proteins in Plants, Production of Therapeutic Proteins in Plants. University of California, Agriculture and Natural Resources. doi: 10.3733/ucanr.8078.
Wibowo, M.S. (2010). Elektroforesis. Bandung: Sekolah Farmasi Institut Teknologi Bandung.
Wijiyadi, E. (2009). Penerapan tree dalam klasifikasi dan determinasi makhluk hidup. Bandung: ITB.
World Health Organization. (2021). World Health Statistic 2021: Monitoring Health for the SDGs, Sustainable Development Goals. Geneva: WHO, 2021.
Yoon, Seon-Joo., Myeong-Ae, Yu., Gwan-Sub, Sim., Seung-Taek, Kwon., Jae-Kwan, Hwang., Jung-Kue, Shin., Ik-Hyun, Yeo. & Yu-Rang, Pyun. (2002). Screening and characterization of microorganisms with fibrinolytic activity from fermented foods. Journal Microbiol Biotechnol, 12(4), pp. 649–656.