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ANTIMICROBIAL ACTIVITY PRESENT in Ganoderma Curtisii AQUEOUS EXTRACTS Actividad Antimicrobial De Extractos Acuosos De Ganoderma Curtissii

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ANTIMICROBIAL ACTIVITY PRESENT in Ganoderma Curtisii AQUEOUS EXTRACTS Actividad Antimicrobial De Extractos Acuosos De Ganoderma Curtissii ANTIMICROBIAL ACTIVITY PRESENT IN Ganoderma curtisii AQUEOUS EXTRACTS Actividad antimicrobial de extractos acuosos de Ganoderma curtissii Alfonso Enrique Islas Rodríguez1, Dolores Marina Barragán Reynaga1, Carlos Álvarez Moya1, Mónica Reynoso Silva1, Laura Guzmán Dávalos2 y Alma Rosa Villaobos Arámbula1* Universidad de Guadalajara, Centro Universitario de Ciencias Biológicas y Agropecuarias. 1Depto. de Biología Celular y Molecular . 2 Depto. de Botánica y Zoología. Camino Ing. Ramón Padilla No. 2100. La venta del Astillero, Zapopan, 45110, Jalisco México. *Corresponding author: [email protected] Abstract extracts show peptide bands measuring approximately 5 kDa, along with bands Macroscopic mushrooms have proven to measuring between 10 and 15 kDa, which be excellent sources of protein. In could represent the antimicrobial peptides addition to this, their medicinal properties that completely inhibited the growth of E. have been noted, including their natural coli when the conventional CFU antitumor, antiviral, antimicrobial, and microdilution method was used. A antioxidant action, as well as their role in preliminary conclusion is that the immune system modulation. Although findings reported in this study can be seen polysaccharides and triterpenoids have as potentially useful and original for been identified as the main antimicrobial therapeutic application in the field of components of the Ganoderma species, biomedicine. some studies mention peptidic components that possess antimicrobial Keywords: antimicrobial activity, effects such as ganodermin, a 15-kDa antimicrobial peptides, fungal protein, antifungal peptide. In view of the growing SDS-PAGE, Ganoderma curtisii, demand for molecules with potential Ganodermataceae therapeutic applications that could help fight antibiotic-resistant microorganisms, Resumen the purpose of this work consists of studying the antimicrobial activity of Los hongos macroscópicos se han aqueous extracts of Ganoderma curtisii, utilizado como excelentes fuentes de found in “La Primavera” Forest in Jalisco, proteína. Además se han mencionado sus Mexico. The aqueous extracts of G. propiedades medicinales, tales como curtisii showed specific dose-response antitumorales, antivirales, antibacterial activity against E. coli. antimicrobianas, antioxidantes naturales, Furthermore, PAGE-SDS gels obtained así como agentes inmunomoduladores. from the pileus of the G. curtisii aqueous Aunque se han mencionado polisacáridos JULIO-DICIEMBRE/AÑO 7/2020/NÚMERO 14/18-24 18 ANTIMICROBIAL ACTIVITY PRESENT IN Ganoderma curtisii AQUEOUS EXTRACTS y triterpenoides como los principales mushrooms are excellent sources of componentes antimicrobianos de las protein. Furthermore, popular culture has especies de Ganoderma, algunos estudios made frequent reference to their mencionan componentes peptídicos que medicinal properties. Now, from a poseen efectos antimicrobianos; por scientific perspective, Ganoderma species ejemplo, “ganodermin” un péptido complex have been reported as important antifúngico de 15 kDa. Debido a la sources of antimicrobial bioactive demanda creciente de moléculas con compounds. Besides the major secondary posibles aplicaciones terapéuticas que metabolites (terpenes, terpenoids and puedan ayudar en la lucha contra los polyketides of farnesyl quonines types) microorganismos resistentes a los with anti-tumor, anti-inflammatory, antibióticos convencionales, el objetivo antioxidant, immune-enhancing, and de este trabajo fue estudiar la actividad antimicrobial activities; small peptides, antimicrobiana de extractos acuosos de polysaccharide, and chitosan also possess Ganoderma curtisii, proveniente del antimicrobial and anti-parasitic properties bosque de “La Primavera” en Jalisco, (Basnet et al., 2017). Although extensive México. Los resultados muestran que los researches on antimicrobial bioactive extractos acuosos de G. curtisii compounds have been carried out on exhibieron actividad antibacteriana Ganoderma sp; most of the studies are específica potencial contra E. coli de una focused on few species, Ganoderma manera dosis-respuesta. Además, los lucidum for instance, produces the 15- geles de PAGE-SDS del píleo de kDa anti-fungal peptide, ganodermin, extractos acuosos de G. curtisii muestran which has inhibitory effects against bandas de péptidos de aproximadamente common fungi Botrytis cinerea and 5 kDa, junto con otras bandas de entre 10 Fusarium oxysporum, (Basnet et al., y 15 KDa, y podrían representar los 2017; Wang et al., 2017; Wang y Ng, péptidos antimicrobianos que inhibieron 2006). The European mushroom name, completamente el crecimiento de E. coli Ganoderma lucidum, has been misapplied en el método de microdilución de UFC to this species, reidentified as G. lingzhi convencional. Se puede concluir de (Dai et al., 2017). Ganoderma curtisii is a manera preliminar que los hallazgos closely related species to G. lingzhi based reportados en este estudio, pueden on phylogenetic analysis (Costa-Rezende considerase potencialmente útiles y et al., 2017), and is frequently found in originales para su aplicación terapéutica Mexico (Torres-Torres y Guzmán- en el campo biomédico. Dávalos, 2005; López-Peña et al., 2016). Palabras Clave: actividad antibacteriana, Fungi, in particular Basidiomycota, péptidos antimicrobianos, proteínas are a still underexplored, highly fúngicas, SDS-PAGE, Ganoderma promising source of antimicrobial curtisii, Ganodermataceae peptides. We are currently living in the “post-antibiotic” era, where both, the Introduction numbers and percentages of multi- resistant bacterial and fungal pathogens Macroscopic mushrooms have been a against the established antibiotics and source of food for thousands of years. synthetic antibacterial agents are Due to their low fat content and the drastically increasing, while the number absence of cholesterol, many macroscopic of new therapeutic agents has decreased (Hyde et JULIO-DICIEMBRE/AÑO 7/2020/NÚMERO 14/18-24 19 ALFONSO ENRIQUE ISLAS RODRÍGUEZ, ET AL al., 2019). It is known that macroscopic molecules with potential therapeutic mushrooms contain multiple proteins applications, the purpose of this work with interesting biological activity for consists of studying the antimicrobial biomedical applications such activity of aqueous extracts of G. curtisii antimicrobial qualities (Basnet et al., found in “La Primavera” Forest in Jalisco, 2017). The bioactive proteins present in Mexico. macroscopic mushrooms, however, will need to be isolated and analyzed to then Materials and Methods prove their biomedical potential and their suitability for treating diseases. Fungal material The reports on antimicrobial peptides The basidiocarp of Ganoderma curtisii in Ganoderma species complex are scarce that was used for this study was collected (Antimicrobial peptides data base 3– in July 2016 at a location in “La APD3) and in the G. curtisii, the Primavera” forest, located in the bioactive compounds studies are focused municipality of Zapopan in the Mexican on antioxidant activity of the phenolic state of Jalisco (L. Guzmán-Dávalos and polysaccharides content (Huerta et 11377, IBUG). The specimen was al., 2016); lanostane triterpenoids and initially identified based on macro and their anti-inflammatory activities (Jiao et micro-morphological characteristics of al., 2016); chemical composition of main the basidiome using conventional sterols (Islas-Santillán et al., 2017) and taxonomical techniques, and its neuroprotective potential against epilepsy identification was confirmed based on of soluble polysaccharides (León-Rivera molecular tools using the Internal et al., 2019). Experts around the world are Transcribed Spacer (ITS) region of the now giving warnings about the serious rDNA. consequences that the lack of antibiotics—in particular against the Preparation of the fungal extracts multi-resistant Gram negative human pathogenic bacteria—can have. After two Samples of the fresh fruiting body decades of neglect, efforts of both the (pileus and stipe) were homogenized in a private and the academic sector on the PBS pH 7,2 (1ml/mg) buffer solution. discovery of new antibiotics have After centrifugation (10.000 X g. 30 substantially increased (Hyde et al., minutes) the supernatant was used to 2019). The Ganoderma sp. medicinal analyze antimicrobial activity and mushroom is considered a key source of electrophoretic band proteins. therapeutic agents to treat infectious bacteria, viruses and parasites (Basnet et Protein determination al., 2017). This study shows the antimicrobial properties of G. curtisii Soluble protein content was collected in the protected natural area of determined by the Bradford method “La Primavera, Jalisco”. (Bradford, 1976) using a Bio-Rad protein assay reagent (Bio-Rad, USA). Bovine Despite the limited studies on the serum albumin (BSA) was used as a activities of antimicrobial peptides in the standard protein. Ganoderma species complex and in response to an increasing demand for Determination of antimicrobial activity JULIO-DICIEMBRE/AÑO 7/2020/NÚMERO 14/18-24 20 ANTIMICROBIAL ACTIVITY PRESENT IN Ganoderma curtisii AQUEOUS EXTRACTS The antimicrobial effect of the G. curtisii extract on Staphylococcus aureus (ATCC® 6538 ™), Escherichia coli (ATCC® 9637 ™) and Candida albicans (obtained from the Dermatological Institute of Jalisco) was assessed using the conventional micro-dilution method (Dalgaard et al., 1994) in nine bioassays. The minimum inhibiting concentration Figure 1. Bar graph of the average values obtained
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