Looking for natural antifungal agent: Sensitive assey to detect fungal endo-1,3-β-glucanase inhibitors from crude plant extracts

Irasema Vargas-Arispuro, Marisol Fraijo-Martínez, Socorro Vallejo-Cohen, Consuelo Corrales-Maldonado, Miguel Ángel Martínez-Téllez

Abstract


1,3-β-glucanase is an enzyme involved in fungal cell wall construction, division septum deposition and ascospore wall assembly. Hence, the 1,3-β-glucanase is a target site to develop into new generations of natural antifungal agents. With the aim of seeking antifungal compounds in plant extracts, we implemented a simple and sensitive assay to detect inhibitors of endo-1,3-β-glucanase from crude plant extracts. The assay, especially useful for screen large number of crude plant extracts, is based on the diffusion of glucanase enzyme from wells through agarosa gel containing the enzyme substrate (laminarin). As the substrate is depolymerized, glucanase diffuses outward in a radial manner leaving a circular zone around the well, unstained by dye calcofluor. A linear relationship between the diameter of hydrolyzed zone and pure enzyme concentration, provide the basis for the method. Results of the assay using plant extracts obtained in methanol or dichloromethane are discussing.


Keywords


Natural fungicide; fungal target site; plant extracts; hydrolase

Full Text:

PDF (Español)

References


Baladrón V, Ufano S, Dueñas E, Martín-Cuadrado B, Del Rey F and Vazquez de Aldana C. 2002. Eng1p, an endo-1,3- β-1,3-glucanase localized at the daughter side of cell separation in Sacharomyces cerevisiae. Eukaryot Cell 1:774-786. doi: 10.1128/EC.1.5.774-786.2002

Dooley H, Shaw MW, Spink J and Kildea S. 2016. The effect of succinate dehydrogenase inhibitor/azole mixtures on selection of Zymoseptoria tritici isolates with reduced sensitivity. Pest Management Science 72:1150-1159. doi:10.1002/ps.4093

Douglas, A.M. 2001. Fungal β(1,3)-D-glucan synthesis. Medical Mycilogy 39 Suppl. 1:55-66. Available online: www.tandfonline.com/doi/abs/10.1080/mmy.39.1.55.66.

Gastebois A, Mouyna I, Simenel C, Clavaud C, Coddeville B, Delepierre M, Latge JP and Fontaine T. 2010. Characterization of a new β-(1–3)-Glucan branching activity of Aspergillus fumigatus. Journal of Biological Chemistry 285:2386–2396. doi:10.1074/jbc.M109.077545

Hartl L, Gastebois A, Aimanianda V and Latgé J P. 2011. Characterization of the GPI-anchored Endo- β-(1-3)-Glucanase Eng2 of Aspergillus fumigatus. Fungal Genetics and Biological 48:185-191. doi:10.1016/j.fgb.2010.06.011

Kurtz, MB and Rex JH. 2001. Glucan synthase inhibitors as antifungal agents. Advances in Protein Chemistry 56:423-475. doi:10.1016/S0065-3233(01)56011-8

Liu J and Balasubramanian M. 2001. 1,3-β-glucan synthase: A useful target for antifungal drugs. Current Drug Targets-Infectious Disorders 1:159-169. doi:http://dx.doi.org/10.2174/1568005014606107

Martín-Cuadrado AB, Dueñas E, Sipiczki M, Vazquez de Aldana C R and Del Rey F. 2003. The endo-ß-1,3-glucanase eng1p is required for dissolution of the primary septum during cell separation in Schizosaccharomyces pombe. Journal of Cell Science 116:1689-1698. doi:10.1242/jcs.00377

Mishra SN, Rao Ch V, Ojha SK, Vijayakumar M and Verma A. 2010. An analytical review of plants for anti diabetic activity with their phytoconstituent and mechanism of action. International Journal of Pharmaceutical Sciences and Research 1:29-46. doi: http://dx.doi.org/10.13040/IJPSR.0975-8232.1(1).29-46

Muller, U. 2002. Chemical crop protection research. Methods and challenges. Pure and Applied Chemistry 74:2241-2246. doi: 10.1351/pac200274122241

Nelson, N. A. 1944. Photometric adaptation of the Somogyi method for the determination of glucose. Journal of Biological Chemistry, 153:375-380. Available online: http://www.jbc.org/content/153/2/375.citation.full.html#ref-list-1

Pan SQ, Ye XS and Kuc J. 1991. A technique for detection of chitinase, β-1,3-glucanase, and protein patterns after a single separation using polyacrylamine gel electrophoresis or isoelectofocusing. Phytopathology 81:970-974. Available online: http://www.apsnet.org/publications/phytopathology/backissues/Documents/1991Articles/Phyto81n09_970.pdf

Rivera-Castañeda G, Martínez-Téllez MA, Vallejo-Cohen S, Vargas-ArispuroI, Moya-Sanz P and Primo-Yúfera E.2001. In vitro Inhibition of Mycelial Growth of Tilletia indica by Extracts of Native Plants from Sonora, Mexico. Revista Mexicana de Fitopatologìa 19:2014-217. Available online: ww.socmexfito.org/images/stories/revista_smf/2001/002/61219213.pdf.

Somogyi, M. 1952. Notes in sugar determination. Journal of Biological Chemistry 195: 19-23. Available online: http://www.jbc.org/content/195/1/19.full.pdf

Thompson G, Duton R and Sparks T. 2000. Spinosad-a case atudy: an example from a natural products discovery programme. Pest Managment Science 56:696-702. doi:10.1002/1526-4998(200008)56:8<696::AID-PS182>3.0.CO;2-5

Vargas-Arispuro I, Contreras-Valenzuela A and Martinez-Tellez, MA. 2009. Lignans from Larrea tridentata (Creosote Bush) as fungal β-1,3-glucanase inhibitors. Pesticide Biochemistry and Physiology 94:60-63. doi:10.1016/j.pestbp.2009.04.002

Vargas-Arispuro I, Reyes-Baez R, Rivera-Castañeda G, Martinez-Téllez MA, Rivero-Espejel I. 2005. Antifungal lignans from Creosote Bush (Larrea tridentata). Industrial Crops and Products 22:101-107. doi:10.1016/j.indcrop.2004.06.003




DOI: http://dx.doi.org/10.18781/R.MEX.FIT.1606-5

Refbacks

  • There are currently no refbacks.