Potent Anti-Candida Fraction Isolated from Capsicum chinense Fruits Contains an Antimicrobial Peptide That is Similar to Plant Defensin and is Able to Inhibit the Activity of Different α-Amylase Enzymes


Antimicrobial peptides (AMPs) are molecules present in several life forms, possess broad-spectrum of inhibitory activity against pathogenic microorganisms, and are a promising alternative to combat the multidrug resistant pathogens. The aim of this work was to identify and characterize AMPs from Capsicum chinense fruits and to evaluate their inhibitory activities against yeasts of the genus Candida and α-amylases. Initially, after protein extraction from fruits, the extract was submitted to anion exchange chromatography resulting two fractions. Fraction D1 was further fractionated by molecular exclusion chromatography, and three fractions were obtained. These fractions showed low molecular mass peptides, and in fraction F3, only two protein bands of approximately 6.5 kDa were observed. Through mass spectrometry, we identified that the lowest molecular mass protein band of fraction F3 showed similarity with AMPs from plant defensin family. We named this peptide CcDef3 (Capsicum chinense defensin 3). The antifungal activity of these fractions was analyzed against yeasts of the genus Candida. At 200 μg/mL, fraction F1 inhibited the growth of C. tropicalis by 26%, fraction F2 inhibited 35% of the growth of C. buinensis, and fraction F3 inhibited all tested yeasts, exhibiting greater inhibition activity on the growth of the yeast C. albicans (86%) followed by C. buinensis (69%) and C. tropicalis (21%). Fractions F1 and F2 promoted membrane permeabilization of all tested yeasts and increased the endogenous induction of reactive oxygen species (ROS) in C. buinensis and C. tropicalis, respectively. We also observed that fraction F3 at a concentration of 50 µg/mL inhibited the α-amylase activities of Tenebrio molitor larvae by 96% and human salivary by 100%. Thus, our results show that fraction F3, which contains CcDef3, is a very promising protein fraction because it has antifungal potential and is able to inhibit the activity of different α-amylase enzymes.

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  1. 1.

    Pennisi E (2010) Armed and dangerous. Science 327(5967):804–805. https://doi.org/10.1126/science.327.5967.804

    CAS  Article  PubMed  Google Scholar 

  2. 2.

    Parisi K, Shafee TMA, Quimbar P et al (2019) The evolution, function and mechanisms of action for plant defensins. Semin Cell Dev Biol 88:107–118. https://doi.org/10.1016/j.semcdb.2018.02.004

    CAS  Article  PubMed  Google Scholar 

  3. 3.

    Bongomin F, Gago S, Oladele RO, Denning DW (2017) Global and multi-national prevalence of fungal diseases-estimate precision. J Fungi 3:57. https://doi.org/10.3390/jof3040057

    Article  Google Scholar 

  4. 4.

    Ullivarri MF, Arbulu S, Garcia-Gutierrez E, Cotter PD (2020) Antifungal peptides as therapeutic agents. Front Cell Infect Microbiol 10:105. https://doi.org/10.3389/fcimb.2020.00105

    CAS  Article  Google Scholar 

  5. 5.

    Oliveira JTA, Souza PFN, Vasconcelos IM et al (2019) Mo-CBP3-PepI, Mo-CBP3-PepII, and Mo-CBP3-PepIII are synthetic antimicrobial peptides active against human pathogens by stimulating ROS generation and increasing plasma membrane permeability. Biochimie 157:10–21. https://doi.org/10.1016/j.biochi.2018.10.016

    CAS  Article  PubMed  Google Scholar 

  6. 6.

    Galdiero E, Lombardi L, Falanga A et al (2019) Biofilms: novel strategies based on antimicrobial peptides. Pharmaceutics 11(7):322. https://doi.org/10.3390/pharmaceutics11070322

    CAS  Article  PubMed Central  Google Scholar 

  7. 7.

    Cools TL, Vriens K, Struyfs C et al (2017) The antifungal plant defensin HsAFP1 is a phosphatidic acid-interacting peptide inducing membrane permeabilization. Front Microbiol 8:2295. https://doi.org/10.3389/fmicb.2017.02295

    Article  PubMed  PubMed Central  Google Scholar 

  8. 8.

    Nicola AM, Albuquerque P, Paes HC et al (2019) Antifungal drugs: new insights in research e development. Pharmacol Ther 195:21–38. https://doi.org/10.1016/j.pharmthera.2018.10.008

    CAS  Article  PubMed  Google Scholar 

  9. 9.

    Koehbach J, Craik DJ (2019) The vast structural diversity of antimicrobial peptides. Trends Pharmacol Sci 40:517–528. https://doi.org/10.1016/j.tips.2019.04.012

    CAS  Article  PubMed  Google Scholar 

  10. 10.

    Patel S, Akhtar N (2017) Antimicrobial peptides (AMPs): the quintessential ‘offense and defense’ molecules are more than antimicrobials. Biomed Pharmacother 95:1276–1283. https://doi.org/10.1016/j.biopha.2017.09.042

    CAS  Article  PubMed  Google Scholar 

  11. 11.

    da Cunha NB, Cobacho NB, Viana JFC et al (2017) The next generation of antimicrobial peptides (AMPs) as molecular therapeutic tools for the treatment of diseases with social and economic impacts. Drug Discov Today 22:234–248. https://doi.org/10.1016/j.drudis.2016.10.017

    CAS  Article  PubMed  Google Scholar 

  12. 12.

    Watkins RR, Bonomo RA (2016) Overview: global and local impact of antibiotic resistance. Infect Dis Clin North Am 30:313–322. https://doi.org/10.1016/j.idc.2016.02.001

    Article  PubMed  Google Scholar 

  13. 13.

    Andrade EKV, Rodrigues R, Bard GCV et al (2020) Identification, biochemical characterization and biological role of defense proteins from common bean genotypes seeds in response to Callosobruchus maculatus infestation. J Stored Prod Res 87:101580. https://doi.org/10.1016/j.jspr.2020.101580

    Article  Google Scholar 

  14. 14.

    Guillén-Chable F, Arenas-Sosa I, Islas-Flores I et al (2017) Antibacterial activity and phospholipid recognition of the recombinant defensin J1–1 from Capsicum genus. Protein Expr Purif 136:45–51. https://doi.org/10.1016/j.pep.2017.06.007

    CAS  Article  PubMed  Google Scholar 

  15. 15.

    Pereira LS, Nascimento VV, Ribeiro SFF et al (2018) Characterization of Capsicum annuum L. leaf and root antimicrobial peptides: antimicrobial activity against phytopathogenic microorganisms. Acta Physiol Plant 40:107. https://doi.org/10.1007/s11738-018-2685-9

    CAS  Article  Google Scholar 

  16. 16.

    Taveira GB, Carvalho AO, Rodrigues R et al (2016) Thionin-like peptide from Capsicum annuum fruits: mechanism of action and synergism with fluconazole against Candida species. BMC Microbiol 16:12. https://doi.org/10.1186/s12866-016-0626-6

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  17. 17.

    Maracahipes AC, Taveira GB, Sousa-Machado GBLY et al (2019) Characterization and antifungal activity of a plant peptide expressed in the interaction between Capsicum annuum fruits and the anthracnose fungus. Biosci Rep 39:12. https://doi.org/10.1042/BSR20192803

    Article  Google Scholar 

  18. 18.

    Afroz M, Akter S, Ahmed A et al (2020) Ethnobotany and antimicrobial peptides from plants of the Solanaceae family: an update and future prospects. Front Pharmacol 11:565. https://doi.org/10.3389/fphar.2020.00565

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  19. 19.

    Taveira GB, Da Motta OV, Machado OLT et al (2014) Thionin-like peptides from Capsicum annuum fruits with high activity against human pathogenic bacteria and yeasts. Biopolym - Pept Sci Sect 102:30–39. https://doi.org/10.1002/bip.22351

    CAS  Article  Google Scholar 

  20. 20.

    Schägger H, von Jagow G (1987) Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem 166:368–379. https://doi.org/10.1016/0003-2697(87)90587-2

    Article  PubMed  Google Scholar 

  21. 21.

    Shevchenko A, Tomas H, Havliš J et al (2007) In-gel digestion for mass spectrometric characterization of proteins and proteomes. Nat Protoc 1:2856–2860. https://doi.org/10.1038/nprot.2006.468

    CAS  Article  Google Scholar 

  22. 22.

    Carneiro RF, De Melo AA, De Almeida AS et al (2013) H-3, a new lectin from the marine sponge Haliclona caerulea: purification and mass spectrometric characterization. Int J Biochem Cell Biol 45:2864–2873. https://doi.org/10.1016/j.biocel.2013.10.005

    CAS  Article  PubMed  Google Scholar 

  23. 23.

    Broekaert WF, Terras FRG, Cammue BPA, Vanderleyden J (1990) An automated quantitative assay for fungal growth inhibition. FEMS Microbiol Lett 69:55–59. https://doi.org/10.1016/0378-1097(90)90412-J

    CAS  Article  Google Scholar 

  24. 24.

    Thevissen K, Terras FRG, Broekaert WF (1999) Permeabilization of fungal membranes by plant defensins inhibits fungal growth. Appl Environ Microbiol 65:5451–5458. https://doi.org/10.1128/aem.65.12.5451-5458.1999

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  25. 25.

    Mello EO, Ribeiro SFF, Carvalho AO et al (2011) Antifungal activity of PvD1 defensin involves plasma membrane permeabilization, inhibition of medium acidification, and induction of ROS in fungi cells. Curr Microbiol 62:1209–1217. https://doi.org/10.1007/s00284-010-9847-3

    CAS  Article  PubMed  Google Scholar 

  26. 26.

    Silva FCV, Nascimento VV, Fernandes KV et al (2018) Recombinant production and α-amylase inhibitory activity of the lipid transfer protein from Vigna unguiculata (L. Walp.) seeds. Process Biochem 65:205–212. https://doi.org/10.1016/j.procbio.2017.10.018

    CAS  Article  Google Scholar 

  27. 27.

    Smith PK, Krohn RI, Hermanson GT et al (1985) Measurement of protein using bicinchoninic acid. Anal Biochem 150:76–85. https://doi.org/10.1016/0003-2697(85)90442-7

    CAS  Article  PubMed  Google Scholar 

  28. 28.

    Campos ML, Souza CM, Oliveira KBS et al (2018) The role of antimicrobial peptides in plant immunity. J Exp Bot 69:4997–5011. https://doi.org/10.1093/jxb/ery294

    CAS  Article  PubMed  Google Scholar 

  29. 29.

    Diz MS, Carvalho AO, Ribeiro SFF et al (2011) Characterisation, immunolocalisation and antifungal activity of a lipid transfer protein from chili pepper (Capsicum annuum) seeds with novel α-amylase inhibitory properties. Physiol Plant 142:233–246. https://doi.org/10.1111/j.1399-3054.2011.01464.x

    CAS  Article  PubMed  Google Scholar 

  30. 30.

    Ribeiro SFF, Taveira GB, Carvalho AO et al (2012) Antifungal and other biological activities of two 2S albumin-homologous proteins against pathogenic fungi. Protein J 31:59–67. https://doi.org/10.1007/s10930-011-9375-4

    CAS  Article  PubMed  Google Scholar 

  31. 31.

    Ribeiro SFF, Silva MS, Da Cunha M et al (2012) Capsicum annuum L. trypsin inhibitor as a template scaffold for new drug development against pathogenic yeast. Anton Leeuw Int J G 101(3):657–670. https://doi.org/10.1007/s10482-011-9683-x

    CAS  Article  Google Scholar 

  32. 32.

    Nascimento VV, Mello EO, Carvalho EOLP et al (2015) PvD1 defensin, a plant antimicrobial peptide with inhibitory activity against Leishmania amazonensis. Biosci Rep 35:5. https://doi.org/10.1042/BSR20150060

  33. 33.

    Bard GCV, Zottich U, Souza TAM et al (2016) Purification, biochemical characterization, and antimicrobial activity of a new lipid transfer protein from Coffea canephora seeds. Genet Mol Res 15:4. https://doi.org/10.4238/gmr15048859

    CAS  Article  Google Scholar 

  34. 34.

    Gebara RS, Taveira GB, Santos LA et al (2020) Identification and characterization of two defensins from Capsicum annuum fruits that exhibit antimicrobial activity. Probiotics Antimicrob Proteins 12(3):1253–1265. https://doi.org/10.1007/s12602-020-09647-6

    CAS  Article  Google Scholar 

  35. 35.

    Meyer B, Houlné G, Pozueta-Romero J et al (1996) Fruit-specific expression of a defensin-type gene family in bell pepper. Upregulation during ripening and upon wounding. Plant Physiol 112:615–622. https://doi.org/10.1104/pp.112.2.615

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  36. 36.

    Seo HH, Park S, Park S et al (2014) Overexpression of a defensin enhances resistance to a fruit-specific anthracnose fungus in pepper. PLoS ONE 9:5. https://doi.org/10.1371/journal.pone.0097936

    CAS  Article  Google Scholar 

  37. 37.

    Dias GB, Gomes VM, Zottich UP et al (2013) Isolation, characterization and antifungal activity of proteinase inhibitors from Capsicum chinense Jacq. seeds. Protein J 32:15–26. https://doi.org/10.1007/s10930-012-9456-z

    CAS  Article  PubMed  Google Scholar 

  38. 38.

    Corrêa JAF, Evangelista AG, de Nazareth T, M, Luciano FB, (2019) Fundamentals on the molecular mechanism of action of antimicrobial peptides. Materialia 8:100494. https://doi.org/10.1016/j.mtla.2019.100494

    CAS  Article  Google Scholar 

  39. 39.

    Cotabarren J, Lufrano D, Parisi MG, Obregón WD (2020) Biotechnological, biomedical, and agronomical applications of plant protease inhibitors with high stability: A systematic review. Plant Sci 292:110398. https://doi.org/10.1016/j.plantsci.2019.110398

    CAS  Article  PubMed  Google Scholar 

  40. 40.

    Kosikowska P, Lesner A (2016) Antimicrobial peptides (AMPs) as drug candidates: a patent review (2003–2015). Expert Opin Ther Pat 26:689–702. https://doi.org/10.1080/13543776.2016.1176149

    CAS  Article  PubMed  Google Scholar 

  41. 41.

    Silva MS, Ribeiro SFF, Taveira GB et al (2017) Application and bioactive properties of CaTI, a trypsin inhibitor from Capsicum annuum seeds: membrane permeabilization, oxidative stress and intracellular target in phytopathogenic fungi cells. J Sci Food Agric 97:3790–3801. https://doi.org/10.1002/jsfa.8243

    CAS  Article  PubMed  Google Scholar 

  42. 42.

    Almeida LHO, Oliveira CFR, Rodrigues MS et al (2020) Adepamycin: design, synthesis and biological properties of a new peptide with antimicrobial properties. Arch Biochem Biophys 691:108487. https://doi.org/10.1016/j.abb.2020.108487

    CAS  Article  PubMed  Google Scholar 

  43. 43.

    Wang K, Dang W, Xie J et al (2015) Antimicrobial peptide protonectin disturbs the membrane integrity and induces ROS production in yeast cells. Biochim Biophys Acta - Biomembr 1848:2365–2373. https://doi.org/10.1016/j.bbamem.2015.07.008

    CAS  Article  Google Scholar 

  44. 44.

    De Coninck B, Cammue BPA, Thevissen K (2013) Modes of antifungal action and in planta functions of plant defensins and defensin-like peptides. Fungal Biol Rev 26:109–120. https://doi.org/10.1016/j.fbr.2012.10.002

    Article  Google Scholar 

  45. 45.

    Soares JR, Melo EJT, da Cunha M et al (2017) Interaction between the plant ApDef1 defensin and Saccharomyces cerevisiae results in yeast death through a cell cycle- and caspase-dependent process occurring via uncontrolled oxidative stress. Biochim Biophys Acta - Gen Subj 1861:3429–3443. https://doi.org/10.1016/j.bbagen.2016.09.005

    CAS  Article  PubMed  Google Scholar 

  46. 46.

    Mello EO, Taveira GB, Carvalho AO, Gomes VM (2019) Improved smallest peptides based on positive charge increase of the γ-core motif from D1 and their mechanism of action against Candida species. Int J Nanomedicine 14:407–420. https://doi.org/10.2147/IJN.S187957

    Article  Google Scholar 

  47. 47.

    Moore J, Rajasekaran K, Cary JW, Chlan C (2019) Mode of action of the antimicrobial peptide D4E1 on Aspergillus flavus. Int J Pept Res Ther 25:1135–1145. https://doi.org/10.1007/s10989-018-9762-1

    CAS  Article  Google Scholar 

  48. 48.

    Teixeira V, Feio MJ, Bastos M (2012) Role of lipids in the interaction of antimicrobial peptides with membranes. Prog Lipid Res 51:149–177. https://doi.org/10.1016/j.plipres.2011.12.005

    CAS  Article  PubMed  Google Scholar 

  49. 49.

    Shackelford RE, Kaufmann WK, Paules RS (2000) Oxidative stress and cell cycle checkpoint function. Free Radic Biol Med 28:1387–1404. https://doi.org/10.1016/S0891-5849(00)00224-0

    CAS  Article  PubMed  Google Scholar 

  50. 50.

    Santos LA, Taveira GB, Ribeiro SFF et al (2017) Purification and characterization of peptides from Capsicum annuum fruits which are α-amylase inhibitors and exhibit high antimicrobial activity against fungi of agronomic importance. Protein Expr Purif 132:97–107. https://doi.org/10.1016/j.pep.2017.01.013

    CAS  Article  PubMed  Google Scholar 

  51. 51.

    Silva MS, Ribeiro SFF, Taveira GB et al (2018) Partial characterization of a immune-related lipid transfer protein (LTP) and activity against yeasts of medical interest from proteins of Canavalia ensiformes (Jack-bean) seeds. World J Pharm Res 7:1120–1138. https://doi.org/10.20959/wjpr201815-13076

    CAS  Article  Google Scholar 

  52. 52.

    Payan F (2004) Structural basis for the inhibition of mammalian and insect α-amylases by plant protein inhibitors. Biochim Biophys Acta - Proteins Proteomics 1696:171–180. https://doi.org/10.1016/j.bbapap.2003.10.012

    CAS  Article  Google Scholar 

  53. 53.

    Silva SM, Koehnlein EA, Bracht A et al (2014) Inhibition of salivary and pancreatic α-amylases by a pinhão coat (Araucaria angustifolia) extract rich in condensed tannin. Food Res Int 56:1–8. https://doi.org/10.1016/j.foodres.2013.12.004

    CAS  Article  Google Scholar 

  54. 54.

    Feng GH, Richardson M, Chen MS et al (1996) α-Amylase inhibitors from wheat: amino acid sequences and patterns of inhibition of insect and human α-amylases. Insect Biochem Mol Biol 26:419–426. https://doi.org/10.1016/0965-1748(95)00087-9

    CAS  Article  PubMed  Google Scholar 

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This work was performed at the Universidade Estadual do Norte Fluminense Darcy Ribeiro. We are grateful to Souza L.C.D. and Kokis V.M. for general laboratory technical support.


This study was financed by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior—Brasil (CAPES)—Finance Code 001, Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ) (E-26/203.090/2016; E-26/202.132/2015; E-26/202.330/2019), and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).

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Correspondence to Valdirene M. Gomes or Érica O. Mello.

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Aguieiras, M.C.L., Resende, L.M., Souza, T.A.M. et al. Potent Anti-Candida Fraction Isolated from Capsicum chinense Fruits Contains an Antimicrobial Peptide That is Similar to Plant Defensin and is Able to Inhibit the Activity of Different α-Amylase Enzymes. Probiotics & Antimicro. Prot. (2021). https://doi.org/10.1007/s12602-020-09739-3

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  • Plant antimicrobial peptide
  • Antifungal
  • Mechanism of action
  • α-Amylase