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Revista mexicana de fitopatología

versión On-line ISSN 2007-8080versión impresa ISSN 0185-3309

Rev. mex. fitopatol vol.35 no.1 Texcoco ene. 2017 

Phytopathological notes

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

Irasema Vargas-Arispuro1  * 

Marisol Fraijo-Martínez1 

Socorro Vallejo-Cohen1 

Consuelo Corrales-Maldonado1 

Miguel Ángel Martínez-Téllez1 

1Centro de Investigación en Alimentación y Desarrollo, A.C. Carr. A la Victoria Km 0.6, 83304 Hermosillo, Sonora, México.


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.

Key words: Natural fungicide; fungal target site; plant extracts; hydrolase

Most of the available antifungal agrochemicals are losing their effectiveness because of the development of fungi pathogens resistance (Dooley et al., 2016). A potential and widely accepted way to discovery new plant disease control agents is to look for plant-derivatives compounds, which have been showed to improve antifungal activities and reduces toxicity (Mishra et al., 2010).

An important aspect of current fungicide research is the used of suitable and novel target site, in which compounds exerts its fungicide effect, affecting a process that should be essential to the fungal growth and preferably one that is required to maintain cell viability. Amount these, the fungal cell wall has emerged as a major target, since fungal are encased in a carbohydrate-containing wall. In consequence, agents that inhibits the synthesis of the fungal cell wall will be more selective (Liu and Balasubramanian, 2001). Fungal cell wall of ascomycetes, deuteromycetes, basidiomycetes and some oomycetes, are composed of β-glucans (Douglas, 2001), that are β-1,3-linked glucose homopolymers, with variable amounts of 1,6-β and 1,4-β-linked glucose side chains and chitin that are responsible for cell rigidity (Gastebois et al., 2010). The dynamic structural reorganization of fungal cell wall requires undergo partial lysis to obtain plasticity during morphological changes as cell growth (branching), cell division and germination in filamentous fungi or the cell separation in yeast, where endo-β-1,3-glucanases play an essential role during such morphogenetic events (Baladrón et al., 2002; Martin-Cuadrado, et al, 2003; Hartl et al., 2011). Therefore a strategy for discovering plant disease control agents would be to looking in plant derivatives endo-β-1,3-glucanase inhibitors as a specific target site of those fungi that their cell wall contained β-glucans, specifically in 1,3-glucan link. There are limited screening methodologies to evaluate in a massive way, plant derivatives and other unusual sources for certain type of biological activity such as fungal enzymes activity (Muller, 2002). On the contrary, in the pharmaceutical industries great advances have been made and thousands of compounds with potential biological activity can be screened in rapid fashion, looking for certain type of biological activity (Kurtz and Rex, 2001).

Our laboratory has been evaluated crude extracts of Sonora Desert plants looking for antifungal plant-derived compound, screening hundreds of crude extract using a traditional antifungal assay (Rivera-Castañeda et al., 2001; Vargas-Arispuro et al., 2005). Considering that some crude extracts have showed antifungal properties against a panel of phytopathogenic fungi, we focused on looking for those plant extract that would selectively inhibit the synthesis of the fungal cell wall, thorough of inhibit endo-β-1,3-glucanase in those fungi that contained it, such as Aspergillus, Penicillum, Tilletia, Botryotinia, Alternaria, Phytophthora, among others (Vargas-Arispuro et al., 2009). In this work, we implemented a simple highthroughput and target-oriented assay for screening of 40 medicinal plant crude extracts for detecting inhibitors to fungal endo-1,3-β-glucanase. This assay would facilitate the biological evaluation of plant crude extracts during the Hit-to-lead transition of a novel class of plant disease control agent discovery.

Forty medicinal/aromatic plants species listed in Table1, were collected from native populations of the Sonora Desert and sun-dried for several weeks. A voucher specimen was deposited at the Herbarium of Universidad de Sonora (Mexico).

Table 1. Evaluation of plan extract obtained in methanol (MeOH) or dichloromethane (DCM) to detect fungal endo-β-1,3-glucanase inhibitors using the gel diffusion assay. 

To plant extracts, five hundred grams of leaves and small stems of dried plants were sequentially extracted with dichloromethane (1 L) and methanol (1 L), the slurry was held at room temperature for 7 days in darkness. The filtrates of each solvent were evaporated to dryness under reduced pressure to obtain a viscous residue, called plant crude extract. The crude extracts were re-suspended in dimetylsulfoxide to perform the gel diffusion assay.

To agarose gel preparation, one point six rams of agarose were dissolved in 100 mL of incubation buffer (0.1 M citric acid, 0.2 M sodium phosphate, pH 5.0) in a microwave oven. The mix was cooled at 50 °C and laminarin from Laminaria digitata (0.5 g in 100 mL of 0.1 M citric acid/0.2 M sodium phosphate buffer, pH 5.0) was gradually added, and the solution was vigorously shaken. Twenty mL of warm agarose-laminarin mix were pipeted into glass Petri dishes (14 cm x 2 cm) and let them cooled at room temperature until agarose solidified. After that, a cork borer was used to cut 2-mm diameter wells in the gel, evenly spaced as radii in the Petri dishes. Excised gel pieces were removed by vacuum.

To Diffusion assay, endo1,3-β-glucanase standard from Trichoderma species (5 unit) was dissolved in 1 mL of 0.1 M citric acid/0.2 M sodium phosphate buffer (pH 5.0) to prepare a standard stock solution. Crude plant extracts (17.5 mL) and pure glucanase (2.5 mL) were pipeted into individual wells in the gel plates. One well with pure glucanase (2.5mL) and incubation buffer (17.5 mL) were added to each Petri dish as a positive control and one well with 20 mL of incubation buffer were added as negative control. The Petri dishes were incubated at 28 °C for 16 h. After incubation, gel were stained with 20 mL of freshly prepared calcofluor [0.1 g in 100 mL of 500 mM Tris-HCl (pH 8.9)] for 10 min. The gels were gently washed three times with distilled water during two hours, using a shaker.

Hydrolyzed laminarin zones in the gel were visualized by UV transillumination as nonfluorescent dark circles in a fluorescent background (undigested laminarin stained by calcofluor). The diameter of hydrolyzed zone were measured (mm) on agarose gel. The assay were conducted at least five times using eight replicates for each extract.

During standardization of gel diffusion assay, laminarin incorporated in agarose gel was easily hydrolyzed by action of endo-1,3-β-glucanase enzyme, produced a clear circles around the wells, which were measurable. Figure 1 shows a linear relationship between the diameters of the hydrolyzed zones in the gel with the units of enzyme added to the well. The coefficient of determination for the regression line was 0.9879. Similar results were obtained in five other experiments. This result suggests that assay can be used for qualitative and quantitative analysis when been necessary.

Figure1. Regression line correlating the diameter of hydrolyzed zones in the plate with unit of pure glucanase. Each point represents the means of 8 replicate samples. Similar regression equations were obtained in five other experiments 

After the assay conditions were established, we evaluated 80 crude plant extracts (40 from dichloromethane and 40 from methanol) in order to detect endo-β-(1-3)-glucanase inhibitors. Figure 2 shows images of gel diffusion agar assay in which dark circles around the wells represent plant extracts that did not contain inhibitors of endoglucanase activity (E3-E11). On the contrary, wells in which no dark circles appeared were considered as plant extracts that contained compounds that inhibit the enzyme activity (E1, E2 and E12).

Figure 2. Images of gel diffusion agar assay to detect inhibitors of β-(1-3)-glucanase in crude plant extracts. Each well label as E contained 17.5 µL of crude plant extract and 2.5 µL of glucanase. Well S contained 2.5 µL (0.125 unit) of pure glucanase from Trichoderma species and 17.5 µL of incubation buffer. Well B contained 20 µL of incubation buffer 

Results of the radial gel diffusion assay to detect inhibitors of fungal endo-β-(1-3)-glucanase activity in crude plant extracts are shown in Table 1. Of the 80 plant extracts evaluated just 3 obtained with metanol (MeOH) and 4 obtained in dichloromethane (DCM), showed endo-1,3-β-glucanase inhibition (Table 1). These results suggest that 7.5 % of MeOH extracts and 10 % of DCM extract, contain compounds that inhibit fungal enzyme. Although other assays to detect glucanase inhibitors may be more sensitive and accurate, they may also be time consuming, and require specialized equipment (Nelson, 1944; Somogyi, 1952) or several steps (Pan et al., 1991).

Natural products screening remains one of the most useful avenues for finding new, unique molecules to serve as leads for the development of novel crop protection agents (Thompson et al., 2000). Developing more rapid throughput screens for natural products should greatly accelerate progress in the discovery of these molecules. Because of limited screening methodologies, often hundreds of natural compounds are discarded. The gel diffusion assay is a valuable tool for screening crude plant extract in a massive, rapid and inexpensive way, compared with Somogyi and Nelson coupled procedure.

Several experiments indicating a high reproducibility of the assay, for this reason, we can concluded that the gel diffusion assay was successfully used to detection of inhibitors of fungal endo- β-(1-3)-glucanase present in crude plant extracts.

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Received: June 20, 2016; Accepted: October 03, 2016

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