Anticancer Activity of Venom Protein Hydrolysis Fraction of Equatorial Spitting Cobra (Naja sumatrana)

Naseer Ahmed(1), Garnis Putri Erlista(2), Tri Joko Raharjo(3*), Respati Tri Swasono(4), Slamet Raharjo(5)

(1) Department of Chemistry, Faculty of Mathematics and Natural Sciences, Universitas Gadjah Mada, Sekip Utara, Yogyakarta 55281, Indonesia
(2) Department of Chemistry, Faculty of Mathematics and Natural Sciences, Universitas Gadjah Mada, Sekip Utara, Yogyakarta 55281, Indonesia
(3) Department of Chemistry, Faculty of Mathematics and Natural Sciences, Universitas Gadjah Mada, Sekip Utara, Yogyakarta 55281, Indonesia
(4) Department of Chemistry, Faculty of Mathematics and Natural Sciences, Universitas Gadjah Mada, Sekip Utara, Yogyakarta 55281, Indonesia
(5) Department Internal Medicine, Faculty of Veterinary Medicine, Universitas Gadjah Mada, Sekip Utara, Yogyakarta 55281, Indonesia
(*) Corresponding Author


Bioactive peptides play an important role in targeting cancer cells. Venom protein from Naja sumatrana can be explored as a source of bioactive peptides. This research aims to identify and study the molecular docking of bioactive peptides (BPs) from trypsin hydrolysate of N. sumatrana venom protein which was fractionated using an SPE C18 column. The venom of N. sumatrana was hydrolyzed with trypsin enzyme. The protein hydrolysate was then fractionated using an RP-SPE HyperSep Retain PEP column, and the peptide fractions were tested for their anticancer activity against MCF-7 breast cancer cells using the (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. Identification of peptides in the active fraction was carried out through high-resolution mass spectrometry. The identified peptides were molecularly docked with the EGFR receptor using AutoDock Vina. The results showed that the degree of hydrolysis was 74.7%. The 75% methanol fraction is the active fraction against MCF-7 cells, with an IC50 value of 4.80 μg/mL and a selectivity index of 5.00. Peptide-active anticancer fractions with the sequence of NSLLVK, SSLLVK and TVPVKR were successfully identified and exhibited high binding affinity values, good RMSD values, and the most suitable model for the epidermal growth factor receptor.


bioactive peptides; trypsin; venom; N. sumatrana; molecular docking

Full Text:

Full Text PDF


[1] Torre, L.A., Bray, F., Siegel, R.L., Ferlay, J., Lortet-Tieulent, J., and Jemal, A., 2015, Global cancer statistics, 2012, Ca-Cancer J. Clin., 65 (2), 87–108.

[2] Fadelu, T., and Rebbeck, T.R., 2021, The rising burden of cancer in low– and middle–Human Development Index countries, Cancer, 127 (16), 2864–2866.

[3] Pérez-Peinado, C., Defaus, S., and Andreu, D., 2020, Hitchhiking with nature: snake venom peptides to fight cancer and superbugs, Toxins, 12 (4), 255.

[4] Nikolaou, M., Pavlopoulou, A., Georgakilas, A.G., and Kyrodimos, E., 2018, The challenge of drug resistance in cancer treatment: A current overview, Clin. Exp. Metastasis, 35 (4), 309–318.

[5] Wang, S.H., and Yu, J., 2018, Structure-based design for binding peptides in anti-cancer therapy, Biomaterials, 156, 1–15.

[6] dos Santos-Silva, C.A., Zupin, L., Oliveira-Lima, M., Vilela, L.M.B., Bezerra-Neto, J.P., Ferreira-Neto, J.R., Ferreira, J.D.C., de Oliveira-Silva, R.L., de Jesús Pires, C., Aburjaile, F.F., de Oliveira, M.F., Kido, E.A., Crovella, S., and Benko-Iseppon, A.M., 2020, Plant antimicrobial peptides: state of the art, in silico prediction and perspectives in the omics era, Bioinf. Biol. Insights, 14, 1177932220952739.

[7] Calderon, L.A., Sobrinho, J.C., Zaqueo, K.D., de Moura, A.A., Grabner, A.N., Mazzi, M.V., Marcussi, S., Nomizo, A., Fernandes, C.F.C., Zuliani, J.P., Carvalho, B.M.A., da Silva, S.L., Stábeli, R.G., and Soares, A.M., 2014, Antitumoral activity of snake venom proteins: New trends in cancer therapy, Biomed Res. Int., 2014, 203639.

[8] Aziz, M.M., Abdel-Rahman, M.A., Abbas, O.A., Mohammed, E.A., and Hassan, M.K., 2022, Snake venoms-based compounds as potential anticancer prodrug: sand viper cerastes as a model, Egypt. J. Chem., 65 (8), 43–55.

[9] Pereira, A., Kerkis, A., Hayashi, M.A.F., Pereira, A.S.P., Silva, F.S., Oliveira, E.B., Prieto da Silva, A.R.B., Yamane, T., Rádis-Baptista, G., and Kerkis, I., 2011, Crotamine toxicity and efficacy in mouse models of melanoma, Expert Opin. Invest. Drugs, 20 (9), 1189–1200.

[10] Nunes, E.S., Souza, M.A.A., Vaz, A.F.M., Silva, T.G., Aguiar, J.S., Batista, A.M., Guerra, M.M.P., Guarnieri, M.C., Coelho, L.C.B.B., and Correia, M.T.S., 2012, Cytotoxic effect and apoptosis induction by Bothrops leucurus venom lectin on tumor cell lines, Toxicon, 59 (7-8), 667–671.

[11] de Vasconcelos Azevedo, F.V.P., Zóia, M.A.P., Lopes, D.S., Gimenes, S.N, Vecchi, L., Alves P.T., Rodrigues, R.S., Silva, A.C.A., Yoneyama, K.A.G., Goulart, L.R., and de Melo Rodrigues, V., 2019, Antitumor and antimetastatic effects of PLA2-BthTX-II from Bothrops jararacussu venom on human breast cancer cells, Int. J. Biol. Macromol., 135, 261–273.

[12] Kerkis, I., Hayashi, M.A.F., Prieto Da Silva, A.R.B., Pereira, A., De Sá Júnior, P.L., Zaharenko, A.J., Rádis-Baptista, G., Kerkis, A., and Yamane, T., 2014, State of the art in the studies on crotamine, a cell penetrating peptide from South American rattlesnake, Biomed Res. Int., 2014, 675985.

[13] Rizzello, C.G., Lorusso, A., Russo, V., Pinto, D., Marzani, B., and Gobbetti, M., 2017, Improving the antioxidant properties of quinoa flour through fermentation with selected autochthonous lactic acid bacteria, Int. J. Food Microbiol., 241, 252–261.

[14] Bhat, Z.F., Kumar, S., and Bhat, H.F., 2015, Bioactive peptides from egg: A review, Nutr. Food Sci., 45 (2), 190–212.

[15] Baharuddin, N.A., Halim, N.R.A., and Sarbon, N.M., 2016, Effect of degree of hydrolysis (DH) on the functional properties and angiotensin I-converting enzyme (ACE) inhibitory activity of eel (Monopterus sp.) protein hydrolysate, Int. Food Res. J., 23 (4), 1424–1431.

[16] Halim, N.R.A., Yusof, H.M., and Sarbon, N.M., 2016, Functional and bioactive properties of fish protein hydrolysates and peptides: A comprehensive review, Trends Food Sci. Technol., 51, 24–33.

[17] Halim, N.R.A., Azlan, A., Yusof, H.M., and Sarbon, N.M., 2018, Antioxidant and anticancer activities of enzymatic eel (Monopterus sp.) protein hydrolysate as influenced by different molecular weight, Biocatal. Agric. Biotechnol., 16, 10–16.

[18] Umayaparvathi, S., Meenakshi, S., Vimalraj, V., Arumugam, M., Sivagami, G., and Balasubramanian, T., 2014, Antioxidant activity and anticancer effect of bioactive peptide from enzymatic hydrolysate of oyster (Saccostrea cucullata), Biomed. Prev. Nutr., 4 (3), 343–353.

[19] Hoskin, D.W., and Ramamoorthy, A., 2008, Studies on anticancer activities of antimicrobial peptides, Biochim. Biophys. Acta, Biomembr., 1778 (2), 357–375.

[20] Kaur, K., Kaur, P., Mittal, A., Nayak, S.K., and Khatik, G.L., 2017, Design and molecular docking studies of novel antimicrobial peptides using AutoDock molecular docking software, Asian J. Pharm. Clin. Res., 10 (16), 28–31.

[21] Yap, M.K.K., Fung, S.Y., Tan, K.Y., and Tan, N.H., 2014, Proteomic characterization of venom of the medically important Southeast Asian Naja sumatrana (Equatorial spitting cobra), Acta Trop., 133, 15–25.

[22] Tan, C.H., Tan, K.Y., Wong, K.Y., Tan, N.H., and Chong, H.P., 2022, Equatorial spitting cobra (Naja sumatrana) from Malaysia (Negeri Sembilan and Penang), Southern Thailand, and Sumatra: Comparative venom proteomics, immunoreactivity and cross-neutralization by antivenom, Toxins, 14 (8), 522.

[23] Gutiérrez, J.M., and Lomonte, B., 2013, Phospholipases A2: Unveiling the secrets of a functionally versatile group of snake venom toxins, Toxicon, 62, 27–39.

[24] Wong, K.Y., Tan, C.H., and Tan, N.H., 2016, Venom and purified toxins of the spectacled cobra (Naja naja) from Pakistan: Insights into toxicity and antivenom neutralization, Am. J. Trop. Med. Hyg., 94 (6), 1392–1399.

[25] Chang, L.S., Huang, H.B., and Lin, S.R., 2000, The multiplicity of cardiotoxins from Naja naja atra (Taiwan cobra) venom, Toxicon, 38 (8), 1065–1076.

[26] Katali, O., Shipingana, L., Nyarangó, P., Pääkkönen, M., Haindongo, E., Rennie, T., James, P., Eriksson, J., and Hunter, C.J., 2020, Protein identification of venoms of the African spitting cobras, Naja mossambica and Naja nigricincta nigricincta, Toxins, 12 (8), 520.

[27] Wong, K.Y., Tan, K.Y., Tan, N.H., and Tan, C.H., 2021, A neurotoxic snake venom without phospholipase A2: Proteomics and cross-neutralization of the venom from Senegalese cobra, Naja senegalensis (Subgenus: Uraeus), Toxins, 13 (1), 60.

[28] Chong, H.P., Tan, K.Y., and Tan, C.H., 2020, Cytotoxicity of snake venoms and cytotoxins from two Southeast Asian cobras (Naja sumatrana, Naja kaouthia): Exploration of anticancer potential, selectivity, and cell death mechanism, Front. Mol. Biosci., 7, 583587.

[29] Silva-de-França, F., Villas-Boas, I.M., de Toledo Serrano, S.M., Cogliati, B., de Andrade Chudzinski, S.A., Lopes, P.H., Kitano, E.S., Okamoto, C.K., and Tambourgi, D.V., 2019, Naja annulifera snake: New insights into the venom components and pathogenesis of envenomation, PLoS Neglected Trop. Dis., 13 (1), e0007017.

[30] Deng, Y., Gruppen, H., and Wierenga, P.A., 2018, Comparison of protein hydrolysis catalyzed by bovine, porcine, and human trypsins, J. Agric. Food Chem., 66 (16), 4219–4232.

[31] Olsen, J.V., Ong, S.E., and Mann, M., 2004, Trypsin cleaves exclusively C-terminal to arginine and lysine residues, Mol. Cell. Proteomics, 3 (6), 608–614.

[32] Butré, C.I., Sforza, S., Gruppen, H., and Wierenga, P.A., 2014, Determination of the influence of substrate concentration on enzyme selectivity using whey protein isolate and Bacillus licheniformis protease, J. Agric. Food Chem., 62 (42), 10230–10239.

[33] Nasri, R., Younes, I., Jridi, M., Trigui, M., Bougatef, A., Nedjar-Arroume, N., Dhulster, P., Nasri, M., and Karra-Châabouni, M., 2013, ACE inhibitory and antioxidative activities of Goby (Zosterissessor ophiocephalus) fish protein hydrolysates: Effect on meat lipid oxidation, Food Res. Int., 54 (1), 552–561.

[34] Raharjo, T.J., Utami, W.M., Fajr, A., Haryadi, W., and Swasono, R.T., 2021, Antibacterial peptides from tryptic hydrolysate of Ricinus communis seed protein fractionated using cation exchange chromatography, Indones. J. Pharm., 32 (1), 74–85.

[35] Atmawati, D.R., Andriana, Z., Swasono, R.T., and Raharjo, T.J., 2022, Antibacterial peptide from solid phase extraction (SPE) fractionation on trypsin hydrolysis of jatropha (Ricinus communis) seed protein acid extract, Rasayan J. Chem., 15 (2), 1288–1295.

[36] Asmi, N., Ahmad, A., Massi, M.N., and Natsir, H., 2019, The potency of protein hydrolysate from epiphytic bacteria associated with brown algae Sargassum sp. as anticancer agents, J. Phys.: Conf. Ser., 1341, 032013.

[37] Rostammiry, L., Saeidiasl, M.R., Safari, R., and Javadian, R., 2017, Optimization of the enzymatic hydrolysis of soy protein isolate by alcalase and trypsin, Biosci., Biotechnol. Res. Asia, 14 (1), 193–200.

[38] Demirgan, R., Karagöz, A., Pekmez, M., Önay-Uçar, E., Artun, F.T., Gürer, Ç., and Mat, A., 2016, In vitro anticancer activity and cytotoxicity of some papaver alkaloids on cancer and normal cell lines, Afr. J. Tradit., Complementary Altern. Med., 13 (3), 22–26.

[39] Cheng, C.Y., and Su, C.C., 2010, Tanshinone IIA may inhibit the growth of small cell lung cancer H146 cells by up-regulating the Bax/Bcl-2 ratio and decreasing mitochondrial membrane potential, Mol. Med. Rep., 3 (4), 645–650.

[40] Khunsap, S., Pakmanee, N., Khow, O., Chanhome, L., Sitprija, V., Suntravat, M., Lucena, S.E., Perez, J.C., and Sanchez, E.E., 2011, Purification of a phospholipase A2 from Daboia russelii siamensis venom with anticancer effects, J. Venom Res., 2, 42–51.

[41] Ahmed, N., Raharjo, S., Swasono, R.T., and Raharjo, T.J., 2022, The antibacterial peptides (AMPS) originated from tryptic hydrolysis of Naja sumatrana venom fractionated using cation exchange chromatography, Rasayan J. Chem., 15 (4), 2642–2653.

[42] Kumar, P.K., and Piramanayagam, S., 2021, Molecular docking analysis of antimicrobial peptides with the CXCL1 protein target for colorectal cancer, Bioinformation, 17 (3), 369–376.

[43] Tsutsui, S., Kataoka, A., Ohno, S., Murakami, S., Kinoshita, J., and Hachitanda, Y., 2002, Prognostic and predictive value of epidermal growth factor receptor in recurrent breast cancer, Clin. Cancer Res., 8 (11), 3454–3460.

[44] Chen, Z., Li, W., Santhanam, R.K., Wang, C., Gao, X., Chen, Y., Wang, C., Xu, L., and Chen, H., 2019, Bioactive peptide with antioxidant and anticancer activities from black soybean [Glycine max (L.) Merr.] byproduct: Isolation, identification and molecular docking study, Eur. Food Res. Technol., 245 (3), 677–689.

[45] Wargasetia, T.L., Ratnawati, H., Widodo, N., and Widyananda, M.H., 2021, Bioinformatics study of sea cucumber peptides as antibreast cancer through inhibiting the activity of overexpressed protein (EGFR, PI3K, AKT1, and CDK4), Cancer Inf., 20, 11769351211031864.


Article Metrics

Abstract views : 2178 | views : 1348

Copyright (c) 2023 Indonesian Journal of Chemistry

Creative Commons License
This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.


Indonesian Journal of Chemistry (ISSN 1411-9420 /e-ISSN 2460-1578) - Chemistry Department, Universitas Gadjah Mada, Indonesia.

Analytics View The Statistics of Indones. J. Chem.