Thrombolytic protease characterization from leaves and fruit flesh of the jernang rattan plant (Daemonorops draco)

Urbanus Yustus Lebuan(1), Roga Florida Kembaren(2*), Merry Meryam Martgrita(3), Cut Rizlani Kholibrina(4)

(1) Bioprocess Engineering Study Program, Faculty of Biotechnology, Institut Teknologi Del, Jalan Sisingamangaraja, Laguboti, Toba 22381, North Sumatera, Indonesia
(2) Bioprocess Engineering Study Program, Faculty of Biotechnology, Institut Teknologi Del, Jalan Sisingamangaraja, Laguboti, Toba 22381, North Sumatera, Indonesia
(3) Bioprocess Engineering Study Program, Faculty of Biotechnology, Institut Teknologi Del, Jalan Sisingamangaraja, Laguboti, Toba 22381, North Sumatera, Indonesia
(4) Pusat Riset Biomassa dan Bioproduk BRIN Kawasan Sains dan Teknologi Dr.(H.C.) Ir.H. Soekarno, Jl.Raya Bogor Km. 46 Cibinong 16911, Indonesia
(*) Corresponding Author


Thrombolytic agents are used for thrombolytic therapy to dissolve blood clots that form in a blood vessel. All currently used thrombolytic agents have unfavorable shortcomings, such as gastrointestinal bleeding, allergic reactions, and thrombolytic agent resistance, treatment for some of which can be quite expensive. As a result, the search for thrombolytic agents derived from plants is currently taking place. Some plants have been discovered to contain protease enzymes with thrombolytic activity; pharmaceuticals derived from plants are believed to be safer. Jernang rattan (Daemonorops draco) is a plant of the Arecaceae family and is known to produce resin. Jernang rattan resin is also known to have antioxidant, antiseptic, antitumor, antimicrobial, and cytotoxic activity, but very limited information on proteolytic activity of the protease from this plant. This research aims to isolate proteases from the leaves and fruit flesh of the rattan jernang plant (D. draco) and to investigate the proteolytic activity of the isolated proteases. The protease was isolated from the leaves and the fruit flesh, and then partially purified by ammonium sulfate precipitation. The radial caseinolytic assay showed that protease in a 60% ammonium sulfate fraction gave a clear zone, with diameters of 1.4 cm and 1.8 cm for the protease isolated from leaves and fruit flesh, respectively. A Folin‐Ciocalteau assay showed that the enzymes isolated were able to hydrolyze casein and release L‐tyrosine, with activity of 0.158 U/mL and 0.174 U/mL for the protease from the leaves and fruit flesh, respectively. A fibrinogenolytic assay showed that the protease from the fruit flesh hydrolyzed the A‐α, B‐β and the γ chain of human fibrinogen, while the protease from the leaves hydrolyzed the A‐α and γ chain. Both proteases were inhibited by 56% by phenylmethylsulfonyl fluoride (PMSF), indicating that the enzymes are serine proteases. Based on the assay results obtained, it can be concluded that proteases isolated from the leaves and fruit flesh have potential as thrombolytic proteases.


Daemonorops draco; Fibrinogenolytic; Protease; Serine protease; Thrombolytic

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Bradford M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of proteindye binding. Anal. Biochem. 72(1-2):248–254. doi:10.1006/abio.1976.9999.

Choi JH, Kim DW, Park SE, Choi BS, Sapkota K, Kim S, Kim SJ. 2014. Novel thrombolytic protease from edible and medicinal plant Aster yomena (Kitam.) Honda with anticoagulant activity: Purification and partial characterization. J. Biosci. Bioeng. 118(4):372–377. doi:10.1016/j.jbiosc.2014.03.004.

Choi JH, Sapkota K, Park SE, Kim S, Kim SJ. 2013. Thrombolytic, anticoagulant and antiplatelet activities of codiase, a bi-functional fibrinolytic enzyme from Codium fragile. Biochimie 95(6):1266–1277. doi:10.1016/j.biochi.2013.01.023.

Chuang EY, Lin KJ, Su FY, Chen HL, Maiti B, Ho YC, Yen TC, Panda N, Sung HW. 2013. Calcium depletion-mediated protease inhibition and apical-junctional- complex disassembly via an EGTA-conjugated carrier for oral insulin delivery. J. Control. Release 169(3):296–305. doi:10.1016/j.jconrel.2012.11.011.

Chung DM, Choi NS, Chun HK, Maeng PJ, Park SB, Kim SH. 2010. A new fibrinolytic enzyme (55kDa) from Allium tuberosum: purification, characterization, and comparison. J. Med. Food 13(6):1532–1536. doi:10.1089/jmf.2010.1144.

Cupp-Enyard C, Aldrich S. 2008. Sigma’s non-specific protease activity assay - Casein as a substrate. J. Vis. Exp. (19):899. doi:10.3791/899.

Da-Yong L, Ting-Ren L. 2019. Drug discoveries from natural resources. J. Prim. Health Care 3:1–8.

Fathimah AN, Wardani AK. 2014. Extraction and characterization of protease enzyme from Moringa Leaves (Moringa oliefera Lamk.). J. Teknol. Pertan. 15(3):191–200.

Gogoi D, Arora N, Kalita B, Sarma R, Islam T, Ghosh SS, Devi R, Mukherjee AK. 2018. Anticoagulant mechanism, pharmacological activity, and assessment of preclinical safety of a novel fibrin(ogen)olytic serine protease from leaves of Leucas indica. Sci. Rep. 8(1):6210. doi:10.1038/s41598-018-24422-y.

Gogoi D, Ramani S, Bhartari S, Chattopadhyay P, Mukherjee AK. 2019. Characterization of active anticoagulant fraction and a fibrin(ogen)olytic serine protease from leaves of Clerodendrum colebrookianum, a traditional ethno-medicinal plant used to reduce hypertension. J. Ethnopharmacol. 243:112099. doi:10.1016/j.jep.2019.112099.

Khavkina LS, Rozenfeld MA, Leonova VB. 1995. Mechanism of inhibition of fibrinolysis and fibrinogenolysis by the end fibrinogen degradation products. Thromb. Res. 78(2):173–187. doi:10.1016/0049- 3848(95)00045-3.

Luzak B, Golanski J, Przygodzki T, Boncler M, Sosnowska D, Oszmianski J, Watala C, Rozalski M. 2016. Extract from spent hop (Humulus lupulus L.) reduces blood platelet aggregation and improves anticoagulant activity of human endothelial cells in vitro. J. Funct. Foods 22:257–269. doi:10.1016/j.jff.2016.01.029.

Matsubara K, Hori K, Matsuura Y, Miyazawa K. 2000. Purification and characterization of a fibrinolytic enzyme and identification of fibrinogen clotting enzyme in a marine green alga, Codium divaricatum. Comp. Biochem. Physiol. - B Biochem. Mol. Biol. 125(1):137–143. doi:10.1016/S0305- 0491(99)00161-3.

Merlyn Keziah S, Subathra Devi C. 2018. Focalization of thrombosis and therapeutic perspectives: A memoir. Orient. Pharm. Exp. Med. 18(4):281–289. doi:10.1007/s13596-018-0331-0.

Patel GK, Kawale AA, Sharma AK. 2012. Purification and physicochemical characterization of a serine protease with fibrinolytic activity from latex of a medicinal herb Euphorbia hirta. Plant Physiol. Biochem. 52:1–6. doi:10.1016/j.plaphy.2011.12.004.

Pepe A, Frey ME, Muñoz F, Fernández MB, Pedraza A, Galbán G, García DN, Daleo GR, Guevara MG. 2016. Fibrin(ogen)olytic and antiplatelet activities of a subtilisin-like protease from Solanum tuberosum (StSBTc-3). Biochimie 125:6– 10. doi:10.1016/j.biochi.2016.03.015.

Rajeswari S, Vidhya R. 2017. Evaluation of in vitro thrombolytic and antiproteinase activities of Wedelia trilobata (Linn.). Innovare J. Life Sci. 5:6–10.

Saksela O. 1981. Radial caseinolysis in agarose: A simple method for detection of plasminogen activator in the presence of inhibitory substances and serum. Anal. Biochem. 111(2):276–282. doi:10.1016/0003- 2697(81)90564-9.

Sharma A, Radha Kishan KV. 2011. Serine protease inhibitor mediated peptide bond re-synthesis in diverse protein molecules. FEBS Lett. 585(21):3465–3470. doi:10.1016/j.febslet.2011.10.004.

Sharmila S, Jeyanthi Rebecca L, Das MP, Saduzzaman M. 2012. Isolation and partial purification of protease from plant leaves. J. Chem. Pharm. Res. 4(8):3808– 3812.

Silva-López RE, Gonçalves RN. 2019. Therapeutic proteases from plants: Biopharmaceuticals with multiple applications. J. Appl. Biotechnol. Bioeng. 6(2):101– 109. doi:10.15406/jabb.2019.06.00180.

Siritapetawee J, Thumanu K, Sojikul P, Thammasirirak S. 2012. A novel serine protease with human fibrino(geno)lytic activities from Artocarpus heterophyllus latex. Biochim. Biophys. Acta - Proteins Proteomics 1824(7):907–912. doi:10.1016/j.bbapap.2012.05.002.

Troncoso FD, Sánchez DA, Ferreira ML. 2022. Production of plant proteases and new biotechnological applications: An updated review. ChemistryOpen 11(3):1–38. doi:10.1002/open.202200017.

Wahyuni WT, Purwanti S, Batubara I. 2018. Antibacterial and antibiofilm activity of Daemonorops draco Resin. Biosaintifika 10(1):1–6. doi:10.15294/biosaintifika.v10i1.13554.

Xia MY, Wang MW, Cui Z, Tashiro SI, Onodera S, Minami M, Ikejima T. 2006. Dracorhodin perchlorate induces apoptosis in HL-60 cells. J. Asian Nat. Prod. Res. 8(4):335–343. doi:10.1080/10286020500035300.

Yi T, Tang Y, Zhang J, Zhao Z, Yang Z, Chen H. 2012. Characterization and determination of six flavonoids in the ethnomedicine ”Dragon’s Blood” by UPLC-PAD-MS. Chem. Cent. J. 6(1):1–7. doi:10.1186/1752-153X-6-116.

Yusnelti, Muhaimin. 2019. Utilization of jernang resin (Daemonorops draco) as the basic material for making liquid wound medicine. In: J. Phys. Conf. Ser., volume 1338. p. 012011. doi:10.1088/1742- 6596/1338/1/012011.

Zhang X, Shuai Y, Tao H, Li C, He L. 2021. Novel method for the quantitative analysis of protease activity: The casein plate method and its applications. ACS Omega 6(5):3675–3680. doi:10.1021/acsomega.0c05192.


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