ANTIMICROBIAL ACTIVITY PRESENT IN 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 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 , has been misapplied en el método de microdilución de UFC to this species, reidentified as G. 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 , 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

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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 ) 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

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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 from nine inhibition-dose response was analyzed using a Colony-Forming bioassays, showing the Colony- Forming Units Unit (CFU) dilution assay (Ong et al., (CFU) of E.coli in contact with the aqueous 2002). extract of the G. curtisii pileus, compared to the control, using different G. curtisii aqueous extract Statistical analysis concentrations. The first three concentrations (0.18, 0.09, 0.045 mg/mL) completely inhibited bacterial growth. The JMP4-type non-parametric median test was used to determine Of the eight concentrations that were significant differences, considering a tested, only the first three (0.18, 0.09, standard error with a confidence level of 0.045 mg/mL) showed strong 0.05. antibacterial activity and inhibited bacterial growth completely. The Electrophoresis testing of fungal extracts following four concentrations had slight activity against bacterial growth, but it The sample was prepared using a was statistically insignificant. S. aureus Laemmil 2X solution with 5% 2- and C. albicans were not inhibited by the mercaptoethanol/extract, 1:1. The dilution evaluated concentrations of G. curtisii was heated at 100°C for 5 minutes [18] extract. The antimicrobial properties of G. and placed at 20°C for 1 minute before curtisii shown in this study are consistent loading. 15 μL of the 1:1 dilution were set with antibacterial peptide fractions from aside, along with 4 μL of the Plus Protein G. lucidum possessed substantial TM Dual Xtra Standard® (BioRad) antibacterial activity against E. coli and marker, using an SDS-PAGE 4-20% Salmonella typhi (Mishra et al., 2018), Mini-PROTEAN® TGXTM Precast Gel Bacillus subtilis, B. cereus, (Bio-Rad) with 1X Tris/Glycine SDS Staphylococus epidermidis, E. coli, Buffer® (BioRad) pH 8.3. The Pseudomonas aeruginosa, except S. electrophoretic testing was conducted aureus (Sa-ard et al., 2015) and our using a HV Power Pac™ (BioRad) power previous laboratory findings, which were source at 120 V, 18 mA for 113 min. The based on a cultivated specimen of a staining process was done using a Ganoderma sp. fruiting body of unknown Coomassie blue R-250 solution (Bollag y origin. The aqueous extracts of that Edelstein, 1991). sample showed consistent antimicrobial properties against Gram-positive bacteria, Results and Discussion such as S. aureus; Gram-negative bacteria, such as E. coli; and yeasts such Aqueous G. curtisii extracts obtained from the pileus showed specific dose- response antibacterial activity against E. coli (Figura 1).

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as C. albicans (data not published). at the campus of the University of China Unlike the findings for Ganoderma sp., in Hong Kong (Wang y Ng, 2006); the the G. curtisii aqueous extracts Lyophyllum (LAP) antifungal protein, specifically inhibited E. coli, and did not with a molecular weight of 14 kDa, inhibit S. aureus or C. albicans. isolated from Lyophyllum shimeji (Ng y Lam, 2002), 16 kDa and 18 kDa proteins Figure 2 shows the G. curtisii aqueous from G. lucidum, both proteins may be extracts in a SDS-PAGE. A strongly the reported lectins and hexameric with stained band of approximately 5 kDa is specific agglutination activity (Li et al., evident in the P lanes, along with other 2018) and 14 kDa protein in the mycelia bands of between 10 and 15 kDa. and fruiting bodies protein extract of G. lucidum (Sa-ard et al., 2015). Of course, it will be necessary to sequence these bands and look for homologies with other closely related species, or determine whether they correspond to new molecules not yet described.

Finally, it is noteworthy that only the pileus extract of the G. curtisii shows electrophoretic bands that could be Figure 2. SDS-PAGE of G. curtisii aqueous related to bioactive molecules, unlike the extracts. M lane, BioRad protein marker; P lanes, stipe extract, which does not show any aqueous pileus extract; S lanes, aqueous stipe such bands (Figure 2). In other cases, extract. specific proteins and peptides, secondary

metabolites and other potent bioactive These bands correspond to the molecules are confined to fruiting bodies aqueous pileus extracts, and could contain and even to specific tissues and different the antimicrobial peptides that completely stages of the mushroom’s development inhibited the growth of E. coli in the (Kües y Badalyan, 2017). A preliminary conventional CFU microdilution method. conclusion is that the findings of this The remaining S lanes correspond to the study can be seen as potentially useful aqueous stipe extract, where no bands and original for therapeutic application in were detected. Interestingly, the strongly the field of biomedicine. stained peptidic bands of low molecular weight of approximately 5 KDa and those between 10 and 15 KDa, which are Acknowledgements shown in PAGE-SDS 4-20% (Figure 2), could correspond to antimicrobial Research supported by PRODEP, PFCE peptides not previously described. and P3e (2016-2018 UDG-CA-44). Curiously, the electrophoretic behavior of the G. curtisii protein bands is similar to References that of ganodermin, the isolated antifungal protein contained in fresh Bradford, M.M. (1976). A rapid and fruiting bodies of the “Ganoderma sensitive method for the quantitation lucidum” medicinal mushroom, collected of microgram quantities of protein utilizing the principle of protein-dye

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