Generic placeholder image

Protein & Peptide Letters


ISSN (Print): 0929-8665
ISSN (Online): 1875-5305

Research Article

Evaluation of the In Vivo Acute Toxicity and In Vitro Hemolytic and Immunomodulatory Activities of the Moringa oleifera Flower Trypsin Inhibitor (MoFTI)

Author(s): Leydianne Leite de Siqueira Patriota, Dayane Kelly Dias do Nascimento Santos, Bárbara Rafaela da Silva Barros, Lethícia Maria de Souza Aguiar, Yasmym Araújo Silva, Angela Caroline Lima Amorim dos Santos, Mariana Gama e Silva, Luana Cassandra Breitenbach Barroso Coelho, Patrícia Maria Guedes Paiva, Emmanuel Viana Pontual, Cristiane Moutinho Lagos de Melo, Rosemairy Luciane Mendes and Thiago Henrique Napoleáo*

Volume 28, Issue 6, 2021

Published on: 13 November, 2020

Page: [665 - 674] Pages: 10

DOI: 10.2174/0929866527999201113105858

Price: $65


Background: Protease inhibitors have been isolated from plants and present several biological activities, including immunomodulatory action.

Objective: This work aimed to evaluate a Moringa oleifera flower trypsin inhibitor (MoFTI) for acute toxicity in mice, hemolytic activity on mice erythrocytes and immunomodulatory effects on mice splenocytes.

Methods: The acute toxicity was evaluated using Swiss female mice that received a single dose of the vehicle control or MoFTI (300 mg/kg, i.p.). Behavioral alterations were observed 15–240 min after administration, and survival, weight gain, and water and food consumption were analyzed daily. Organ weights and hematological parameters were analyzed after 14 days. Hemolytic activity of MoFTI was tested using Swiss female mice erythrocytes. Splenocytes obtained from BALB/c mice were cultured in the absence or presence of MoFTI for the evaluation of cell viability and proliferation. Mitochondrial membrane potential (Δψm) and reactive oxygen species (ROS) levels were also determined. Furthermore, the culture supernatants were analyzed for the presence of cytokines and nitric oxide (NO).

Results: MoFTI did not cause death or any adverse effects on the mice except for abdominal contortions at 15–30 min after administration. MoFTI did not exhibit a significant hemolytic effect. In addition, MoFTI did not induce apoptosis or necrosis in splenocytes and had no effect on cell proliferation. Increases in cytosolic and mitochondrial ROS release, as well as Δψm reduction, were observed in MoFTI-treated cells. MoFTI was observed to induce TNF-α, IFN-γ, IL-6, IL-10, and NO release.

Conclusion: These results contribute to the ongoing evaluation of the antitumor potential of MoFTI and its effects on other immunological targets.

Keywords: Plant protein, protease inhibitor, acute toxicity, hemolytic activity, immunomodulation, cytokines.

Graphical Abstract
Zhu-Salzman, K.; Zeng, R. Insect response to plant defensive protease inhibitors. Annu. Rev. Entomol., 2015, 60, 233-252.
[] [PMID: 25341101]
Dang, L.; Van Damme, E.J.M. Toxic proteins in plants. Phytochemistry, 2015, 117, 51-64.
[] [PMID: 26057229]
Hamza, R.; Pérez-Hedo, M.; Urbaneja, A.; Rambla, J.L.; Granell, A.; Gaddour, K.; Beltrán, J.P.; Cañas, L.A. Expression of two barley proteinase inhibitors in tomato promotes endogenous defensive response and enhances resistance to Tuta absoluta. BMC Plant Biol., 2018, 18(1), 24.
[] [PMID: 29370757]
Cotabarren, J.; Lufrano, D.; Parisi, M.G.; Obregón, W.D. Biotechnological, biomedical, and agronomical applications of plant protease inhibitors with high stability: A systematic review. Plant Sci., 2020, 292, 110398.
[] [PMID: 32005400]
Srikanth, S.; Chen, Z. Plant protease inhibitors in therapeutics-focus on cancer therapy. Front. Pharmacol., 2016, 7, 470.
[] [PMID: 28008315]
Bonturi, C.R.; Motaln, H.; Silva, M.C.C.; Salu, B.R.; de Brito, M.V.; de Andrade Luz Cost, L.; Torquato, H.F.V.; Nunes, N.N.D.S.; Paredes-Gamero, E.J.; Turnšek, T.L.; Oliva, M.L.V. Could a plant derived protein potentiate the anticancer effects of a stem cell in brain cancer? Oncotarget, 2018, 9(30), 21296-21312.
[] [PMID: 29765540]
Bortolozzo, A.S.S.; Rodrigues, A.P.D.; Arantes-Costa, F.M.; Saraiva-Romanholo, B.M.; de Souza, F.C.R.; Brüggemann, T.R.; de Brito, M.V.; Ferreira, R.D.S.; Correia, M.T.D.S.; Paiva, P.M.G.; Prado, C.M.; Leick, E.A.; Oliva, M.L.V.; Martins, M.A.; Ruiz-Schutz, V.C.; Righetti, R.F.; Tibério, I.F.L.C. The plant proteinase inhibitor CrataBL plays a role in controlling asthma response in mice. BioMed Res. Int., 2018, 2018, 9274817.
[] [PMID: 30364003]
Fang, E.F.; Ng, T.B. A trypsin inhibitor from rambutan seeds with antitumor, anti-HIV-1 reverse transcriptase, and nitric oxide-inducing properties. Appl. Biochem. Biotechnol., 2015, 175(8), 3828-3839.
[] [PMID: 25820360]
Patriota, L.L.S.; Procópio, T.F.; de Souza, M.F.; de Oliveira, A.P.; Carvalho, L.V.N.; Pitta, M.G.R.; Rego, M.J.B.M.; Paiva, P.M.G.; Pontual, E.V.; Napoleão, T.H. A trypsin inhibitor from Tecoma stans leaves inhibits growth and promotes ATP depletion and lipid peroxidation in Candida albicans and Candida krusei. Front. Microbiol., 2016, 7, 611.
[] [PMID: 27199940]
Shamsi, T.N.; Fatima, S. Protease inhibitors as ad-hoc antibiotics. Open Pharm. Sci. J., 2016, 3, 131-137.
Pontual, E.V.; Pires-Neto, D.F.; Fraige, K.; Higino, T.M.M.; Carvalho, B.E.A.; Alves, N.M.P.; Lima, T.A.; Zingali, R.B.; Coelho, L.C.B.B.; Bolzani, V.S.; Figueiredo, R.C.B.Q.; Napoleão, T.H.; Paiva, P.M.G. A trypsin inhibitor from Moringa oleifera flower extract is cytotoxic to Trypanosoma cruzi with high selectivity over mammalian cells. Nat. Prod. Res., 2018, 32(24), 2940-2944.
[] [PMID: 29047320]
Tsoi, A.Y.K.; Ng, T.B.; Fong, W.P. Immunomodulatory activity of a chymotrypsin inhibitor from Momordica cochinchinensis seeds. J. Pept. Sci., 2006, 12(9), 605-611.
[] [PMID: 16733830]
Hudson, A.; Lopez, E.; Almalki, A.J.; Roe, A.L.; Calderón, A.I. A review of the toxicity of compounds found in herbal dietary supplements. Planta Med., 2018, 84(9-10), 613-626.
[] [PMID: 29672820]
Krishnan, V.G.M.; Murugan, K. Acute and subchronic toxicological evaluation of the purified protease inhibitor from the fruits of Solanum aculeatissimum Jacq. on Wistar rats. Cogent Biol., 2016, 2, 1191588.
Mayasa, V.; Rasal, V.K.; Unger, B.S.; Subbarayan, K. Tolerability assessment and anti-cancer activity of the partially purified protease inhibitors from Soyabean (Glycine max) orally administered to rats. Res. J. Pharm. Technol., 2016, 9, 925-928.
Ghosh, T.; Biswas, M.K.; Chatterjee, S.; Roy, P. In-vitro study on the hemolytic activity of different extracts of Indian medicinal plant Croton bonplandianum with phytochemical estimation: A new era in drug development. J. Drug Deliv. Ther., 2018, 8, 155-160.
Khalil, D.N.; Smith, E.L.; Brentjens, R.J.; Wolchok, J.D. The future of cancer treatment: immunomodulation, CARs and combination immunotherapy. Nat. Rev. Clin. Oncol., 2016, 13(5), 273-290.
[] [PMID: 26977780]
Matsushita, M.; Kawaguchi, M. Immunomodulatory effects of drugs for effective cancer immunotherapy. J. Oncol., 2018, 2018, 8653489.
[] [PMID: 30498512]
Jantan, I.; Ahmad, W.; Bukhari, S.N.A. Plant-derived immunomodulators: an insight on their preclinical evaluation and clinical trials. Front. Plant Sci., 2015, 6, 655.
[] [PMID: 26379683]
Fura, J.M.; Sarkar, S.; Pidgeon, S.E.; Pires, M.M. Combatting bacterial pathogens with immunomodulation and infection tolerance strategies. Curr. Top. Med. Chem., 2017, 17(3), 290-304.
[] [PMID: 27572083]
Safavi, F.; Rostami, A. Role of serine proteases in inflammation: Bowman-Birk protease inhibitor (BBI) as a potential therapy for autoimmune diseases. Exp. Mol. Pathol., 2012, 93(3), 428-433.
[] [PMID: 23022357]
Malemud, C.J. Immunomodulators in autoimmunity and viral infections. J. Clin. Cell. Immunol., 2018, 9, 537.
Santos, A.J.C.A.; Barros, B.R.S.; Aguiar, L.M.S.; Patriota, L.L.S.; Lima, T.A.; Zingali, R.B.; Paiva, P.M.G.; Napoleão, T.H.; Melo, C.M.L.; Pontual, E.V. Schinus terebinthifolia leaf lectin (SteLL) is an immunomodulatory agent by altering cytokine release by mice splenocytes. 3 Biotech, 2020, 10, 144.
Fang, E.F.; Wong, J.H.; Bah, C.S.F.; Lin, P.; Tsao, S.W.; Ng, T.B. Bauhinia variegata var. variegata trypsin inhibitor: from isolation to potential medicinal applications. Biochem. Biophys. Res. Commun., 2010, 396(4), 806-811.
[] [PMID: 20435016]
Fang, E.F.; Wong, J.H.; Ng, T.B. Thermostable Kunitz trypsin inhibitor with cytokine inducing, antitumor and HIV-1 reverse transcriptase inhibitory activities from Korean large black soybeans. J. Biosci. Bioeng., 2010, 109(3), 211-217.
[] [PMID: 20159565]
de Siqueira Patriota, L.L.; Procópio, T.F.; de Santana Brito, J.; Sebag, V.; de Oliveira, A.P.S.; de Araújo Soares, A.K.; Moreira, L.R.; de Albuquerque Lima, T.; Soares, T.; da Silva, T.D.; Paiva, P.M.G.; de Lorena, V.M.B.; de Melo, C.M.L.; de Albuquerque, L.P.; Napoleão, T.H. Microgramma vacciniifolia (Polypodiaceae) fronds contain a multifunctional lectin with immunomodulatory properties on human cells. Int. J. Biol. Macromol., 2017, 103, 36-46.
[] [PMID: 28501598]
Pramanik, A.; Paik, D.; Pramanik, P.K.; Chakraborti, T. Serine protease inhibitors rich Coccinia grandis (L.) Voigt leaf extract induces protective immune responses in murine visceral leishmaniasis. Biomed. Pharmacother., 2019, 111, 224-235.
[] [PMID: 30584985]
Santos, A.F.S.; Luz, L.A.; Pontual, E.V.; Napoleão, T.H.; Paiva, P.M.G.; Coelho, L.C.B.B. Moringa oleifera: resource management and multiuse life tree. Adv. Res., 2015, 4, 388-402.
Pontual, E.V.; de Lima Santos, N.D.; de Moura, M.C.; Coelho, L.C.B.B.; do Amaral Ferraz Navarro, D.M.; Napoleão, T.H.; Paiva, P.M.G. Trypsin inhibitor from Moringa oleifera flowers interferes with survival and development of Aedes aegypti larvae and kills bacteria inhabitant of larvae midgut. Parasitol. Res., 2014, 113(2), 727-733.
[] [PMID: 24271154]
Lowry, O.H.; Rosebrough, N.J.; Farr, A.L.; Randall, R.J. Protein measurement with the Folin phenol reagent. J. Biol. Chem., 1951, 193(1), 265-275.
[PMID: 14907713]
Kakel, S.J. The evaluation of traditional and automatic Coulter method in estimation of haematological parameters in adult rats. Beni-Suef Univ. J. Basic Appl. Sci., 2013, 2, 31-35.
Pita, J.C.L.L.R.; Xavier, A.L.; de Sousa, T.K.; Mangueira, V.M.; Tavares, J.F.; de Oliveira Júnior, R.J.; Veras, R.C.; Pessoa, Hde.L.; da Silva, M.S.; Morelli, S.; Ávila, Vde.M.; da Silva, T.G.; Diniz, Mde.F.; Castello-Branco, M.V.S. In vitro and in vivo antitumor effect of trachylobane-360, a diterpene from Xylopia langsdorffiana. Molecules, 2012, 17(8), 9573-9589.
[] [PMID: 22885357]
Procópio, T.F.; de Siqueira Patriota, L.L.; da Silva Barros, B.R.; de Souza Aguiar, L.M.; de Lorena, V.M.B.; Paiva, P.M.G.; de Melo, C.M.L.; Napoleão, T.H. Calliandra surinamensis lectin (CasuL) does not impair the functionality of mice splenocytes, promoting cell signaling and cytokine production. Biomed. Pharmacother., 2018, 107, 650-655.
[] [PMID: 30118881]
Ding, A.H.; Nathan, C.F.; Stuehr, D.J. Release of reactive nitrogen intermediates and reactive oxygen intermediates from mouse peritoneal macrophages. Comparison of activating cytokines and evidence for independent production. J. Immunol., 1988, 141(7), 2407-2412.
[PMID: 3139757]
Grigore, A. Plant phenolic compounds as immunomodulatory agents. In: Phenolic Compounds – Biological Activity; Soto-Hernánez, M.; PalmaTenango, M.; Garcia-Mateos, M.R., Eds.; InTech Open: London,, 2017; pp. 75-98.
de Santana Brito, J.; Ferreira, G.R.S.; Klimczak, E.; Gryshuk, L.; de Lima Santos, N.D.; de Siqueira Patriota, L.L.; Moreira, L.R.; Soares, A.K.A.; Barboza, B.R.; Paiva, P.M.G.; do Amaral Ferraz Navarro, D.M.; de Lorena, V.M.B.; de Melo, C.M.L.; Coriolano, M.C.; Napoleão, T.H. Lectin from inflorescences of ornamental crop Alpinia purpurata acts on immune cells to promote Th1 and Th17 responses, nitric oxide release, and lymphocyte activation. Biomed. Pharmacother., 2017, 94, 865-872.
[] [PMID: 28810516]
Liu, L.; Yu, H.; Wu, H.; Yang, X.; Pan, Y.; Chen, Y.; Wang, K.; Wang, W.; Zhang, W.; Jin, Y.; Zhang, C.; Jiang, A.; Xia, C. Toxic proteins from Croton tiglium L. exert a proinflammatory effect by inducing release of proinflammatory cytokines and activating the p38-MAPK signaling pathway. Mol. Med. Rep., 2017, 16(1), 631-638.
[] [PMID: 28560398]
Paula, P.C.; Sousa, D.O.B.; Oliveira, J.T.A.; Carvalho, A.F.U.; Alves, B.G.T.; Pereira, M.L.; Farias, D.F.; Viana, M.P.; Santos, F.A.; Morais, T.C.; Vasconcelos, I.M. A protein isolate from Moringa oleifera leaves has hypoglycemic and antioxidant effects in alloxan-induced diabetic mice. Molecules, 2017, 22(2), 271.
[] [PMID: 28208654]
Coriolano, M.C.; de Santana Brito, J.; de Siqueira Patriota, L.L.; de Araujo Soares, A.K.; de Lorena, V.M.B.; Paiva, P.M.G.; Napoleão, T.H.; Coelho, L.C.B.B.; de Melo, C.M.L. Immunomodulatory effects of the water-soluble lectin from Moringa oleifera seeds (WSMoL) on Human Peripheral Blood Mononuclear Cells (PBMC). Protein Pept. Lett., 2018, 25(3), 295-301.
[] [PMID: 29384049]
de Melo, C.M.; Paim, B.A.; Zecchin, K.G.; Morari, J.; Chiaratti, M.R.; Correia, M.T.S.; Barroso Coelho, L.C.; Paiva, P.M.G. Cramoll 1,4 lectin increases ROS production, calcium levels, and cytokine expression in treated spleen cells of rats. Mol. Cell. Biochem., 2010, 342(1-2), 163-169.
[] [PMID: 20432056]
Previte, D.M.; O’Connor, E.C.; Novak, E.A.; Martins, C.P.; Mollen, K.P.; Piganelli, J.D. Reactive oxygen species are required for driving efficient and sustained aerobic glycolysis during CD4+ T cell activation. PLoS One, 2017, 12(4), e0175549.
[] [PMID: 28426686]
Chen, Y.; Zhou, Z.; Min, W. Mitochondria, oxidative stress and innate immunity. Front. Physiol., 2018, 9, 1487.
[] [PMID: 30405440]
Ježek, P.; Holendová, B.; Plecitá-Hlavatá, L. Redox signaling from mitochondria: signal propagation and its targets. Biomolecules, 2020, 10(1), 93.
[] [PMID: 31935965]
Lee, C.P.; Maksaev, G.; Jensen, G.S.; Murcha, M.W.; Wilson, M.E.; Fricker, M.; Hell, R.; Haswell, E.S.; Millar, A.H.; Sweetlove, L.J. MSL1 is a mechanosensitive ion channel that dissipates mitochondrial membrane potential and maintains redox homeostasis in mitochondria during abiotic stress. Plant J., 2016, 88(5), 809-825.
[] [PMID: 27505616]
Ip, W.K.E.; Hoshi, N.; Shouval, D.S.; Snapper, S.; Medzhitov, R. Anti-inflammatory effect of IL-10 mediated by metabolic reprogramming of macrophages. Science, 2017, 356(6337), 513-519.
[] [PMID: 28473584]
Bertazza, L.; Mocellin, S. The dual role of tumor necrosis factor (TNF) in cancer biology. Curr. Med. Chem., 2010, 17(29), 3337-3352.
[] [PMID: 20712570]
Josephs, S.F.; Ichim, T.E.; Prince, S.M.; Kesari, S.; Marincola, F.M.; Escobedo, A.R.; Jafri, A. Unleashing endogenous TNF-alpha as a cancer immunotherapeutic. J. Transl. Med., 2018, 16(1), 242-249.
[] [PMID: 30170620]
Nie, H.; Zheng, Y.; Li, R.; Guo, T.B.; He, D.; Fang, L.; Liu, X.; Xiao, L.; Chen, X.; Wan, B.; Chin, Y.E.; Zhang, J.Z. Phosphorylation of FOXP3 controls regulatory T cell function and is inhibited by TNF-α in rheumatoid arthritis. Nat. Med., 2013, 19(3), 322-328.
[] [PMID: 23396208]
Dondossola, E.; Dobroff, A.S.; Marchiò, S.; Cardó-Vila, M.; Hosoya, H.; Libutti, S.K.; Corti, A.; Sidman, R.L.; Arap, W.; Pasqualini, R. Self-targeting of TNF-releasing cancer cells in preclinical models of primary and metastatic tumors. Proc. Natl. Acad. Sci. USA, 2016, 113(8), 2223-2228.
[] [PMID: 26858439]
Shadrin, N.; Shapira, M.G.; Khalfin, B.; Uppalapati, L.; Parola, A.H.; Nathan, I. Serine protease inhibitors interact with IFN-γ through up-regulation of FasR; a novel therapeutic strategy against cancer. Exp. Cell Res., 2015, 330(2), 233-239.
[] [PMID: 25449698]
Chonov, D.C.; Ignatova, M.M.K.; Ananiev, J.R.; Gulubova, M.V. IL-6 activities in the tumour microenvironment. Part 1. Open Access Maced. J. Med. Sci., 2019, 7(14), 2391-2398.
[] [PMID: 31592285]
Chuang, M.T.; Lin, Y.S.; Hou, W.C. Ancordin, the major rhizome protein of madeira-vine, with trypsin inhibitory and stimulatory activities in nitric oxide productions. Peptides, 2007, 28(6), 1311-1316.
[] [PMID: 17499881]
Fang, E.F.; Lin, P.; Wong, J.H.; Tsao, S.W.; Ng, T.B. A lectin with anti-HIV-1 reverse transcriptase, antitumor, and nitric oxide inducing activities from seeds of Phaseolus vulgaris cv. extralong autumn purple bean. J. Agric. Food Chem., 2010, 58(4), 2221-2229.
[] [PMID: 20095617]
Bender, D.; Schwarz, G. Nitrite-dependent nitric oxide synthesis by molybdenum enzymes. FEBS Lett., 2018, 592(12), 2126-2139.
[] [PMID: 29749013]

Rights & Permissions Print Cite
© 2024 Bentham Science Publishers | Privacy Policy