Original Articles

The Study of Antimicrobial Activities of Partially Purified Cyclotide Content and Crude Extracts from Viola tricolor


Background:    Searching for the potent natural antibiotics will improve the treatment of most infectious diseases. Cyclotides are potent active peptides derived from some plant families like Violaceae. Viola tricolor (the pansy) has been found to have numerous cyclotides. Nowadays cyclotides attract more attention for their antibacterial activities.  The current research studied the antimicrobial properties of semi-purified cyclotides from V. tricolor. Methods:   Extraction and purification of cyclotides from Iranian Viola tricolor were performed by fractionation and solid phase extraction methods, and their antimicrobial effects were studied against several bacterial strains using diffusion assays. Also, attendance of cyclotide in the extract was verified by Tricine-SDS page and spectroscopic methods. Results:  Antimicrobial effects of semi-purified cyclotides and crude extracts resulted in the antibacterial activity potential of V. tricolor totally extracted samples against gram negative bacteria, E.coli and P. aeruginosa. However, there is need for optimizing the assay method and the culture media. Conclusion:    Viola tricolor as a remedy represents the antibacterial potential, which may not be unrelated to its cyclotide content although the effectiveness of cyclotides may also differ because of their synergism, natural structure and bioactivities, the amount of purified content, and the way they were assayed. 

Livermore DM. The need for new antibiotics. Clin Microbiol Infect 2004; 10: 1-9.

Craik DJ, Daly NL, Bond T, Waine C. Plant cyclotides: a unique family of cyclic and knotted proteins that defines the cyclic cystine knot structural motif. J Mol Biol 1999; 294: 1327-1336.

Jennings C, West J, Waine C, Craik D, and Anderson, M. Biosynthesis and insecticidal properties of plant cyclotides: The cyclic knotted proteins from Oldenlandia affinis. Proc. Natl. Acad. Sci. U.S.A. 2001; 98: 10614-10619.

Lindholm P, Göransson U, Johansson S, Claeson P, Gullbo J, Larsson R., et al. Cyclotides: A novel type of cytotoxic agents. Mol. Cancer Ther 2002; 1: 365-369.

Gustafson KR, Sowder RC, Henderson LE, Parsons IC, Kashman Y, Cardellina JH, et al. Circulins A and B: novel HIV-inhibitory macrocyclic peptides from the tropical tree Chassalia parvifolia. J. Am. Chem. Soc 1994; 116: 9337-9338.

Pränting M, Lööv C, Burman R, Göransson U, Andersson DI. The cyclotide cycloviolacin O2 from Viola odorata has potent bactericidal activity against Gram-negative bacteria. J Antimicrob Chemother 2010; 65: 1964–1971.

Craik DJ, Daly NL, Mulvenna J, Plan MR, Trabi M. Discovery, structure and biological activities of the cyclotides. Curr Protein Pept Sci 2004; 5: 297-315.

Colgrave ML, Craik DJ. Thermal, chemical, and enzymatic stability of the cyclotide kalata B1: the importance of the cyclic cystine knot. Biochemistry 2004; 43: 5965-5975.

Koehbach J, Attah AF, Berger A, Hellinger R, Kutchan TM, et al. Cyclotide discovery in Gentianales revisited-identification and characterization of cyclic cystine-knot peptides and their phylogenetic distribution in Rubiaceae plants. Biopolymers 2013; 100: 438-452.

Svangard E, Goransson U, Hocaoglu Z, Gullbo J, Larsson R, Claeson P, et al. Cytotoxic cyclotides from Viola tricolor. J Nat Prod 2004; 67: 144-7.

Tang J, Wang CK, Pan X, Yan H, Zeng G, Xu W, et al. Isolation and characterization of cytotoxic cyclotides from Viola tricolor. Peptides 2010; 31(8): 1434-1440.

Gruber CW, Elliott AG, Ireland DC, Delprete PG, Dessein S, Göransson U, et al. Distribution and evolution of circular miniproteins in flowering plants. The Plant Cell 2008; 20(9): 2471-2483.

Bisset NG, Wichtl M. Herbal drugs and phytopharmaceuticals. Medpharm. 2nd ed. 1994; p. 527.

Komorowski T, Mosiniak T, Kryszczuk Z, Rosinski G. Phenolic acids in the local species of Viola tricolor L. and Viola arvensis. Herba Pol 1983; 29(1): 5-11.

Witkowska-Banaszczak E, Bylka W, Matlawska I, Goslinska O, Muszynski Z. Antimicrobial activity of Viola tricolor herb. Fitoterapia 2005; 76: 458-61.

Claeson P, Göransson U, Johansson S, Luijendijk T, Bohlin L. Fractionation protocol for the isolation of polypeptides from plant biomass. J Nat Prod 1998; 61: 77-81.

Poth AG, Colgrave ML, Philip R, Kerenga B, Daly NL, et al. Discovery of cyclotides in Fabaceae plant family provides new insight into the cyclization, evolution and distribution of circular proteins. ACS Chem Bio 2011; 6: 345-355.

Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 2011; 72: 248–254.

Schagger H. Tricine-SDS-PAGE. Nat Protoc 2006; 1: 16–23.

Schagger H, Jagow GV. Tricine-sodiom dodecyl sulfate polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem 1987; 166: 368–379.

Lehrer RI, Rosenman M, Harwig SS, Jackson R, Eisenhauer P. Ultrasensitive assays for endogenous antimicrobial polypeptides. J Immunol Methods 1991; 137: 167-173.

Gran L, Sletten K, Skjeldal L. Cyclic peptides from Oldenlandia affinis DC: molecular and biological properties. Chem Biodivers 2008; 5: 2014-2022.

Tam JP, Lu YA, Yang JL, Chiu KW. An unusual structural motif of antimicrobial peptides containing end-to-end macrocycle and cystine-knot disulfides. Proc Natl Acad Sci 1999; 96: 8913-8918.

Zarrabi M, Dalirfardouei R, Sepehrizade Z, Kermanshahi RK. Comparison of the antimicrobial effects of semipurified cyclotides from Iranian Viola odorata against some of plant and human pathogenic bacteria. J App Microbiol 2013; 115: 367-375.

Roshan A, Zarrabi M, Asgarani E, Kanaani MR. Sequence analysis of gene encoding cyclotides in three species of Violaceae and determination of their anti-microbial activities. J Biochem Microb Technol 2014; 2: 1-7.

IssueVol 5 No 1-2 (2016) QRcode
SectionOriginal Articles
Viola tricolor Cyclotide Extraction Antibacterial effect

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How to Cite
Khoshkam Z, Zarrabi M, Sepehrizade Z, Keshavarzi M. The Study of Antimicrobial Activities of Partially Purified Cyclotide Content and Crude Extracts from Viola tricolor. J Med Bacteriol. 2016;5(1-2):29-35.