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Preliminary Bioactivity Screening of Crude Extracts of Six Wild Macrofungi from Pine Forests in Benguet and Mt

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Preliminary Bioactivity Screening of Crude Extracts of Six Wild Macrofungi from Pine Forests in Benguet and Mt Manila Journal of Science 13 (2020), pp. 74–88 Preliminary Bioactivity Screening of Crude Extracts of Six Wild Macrofungi From Pine Forests in Benguet and Mt. Province, Philippines Audrey Glenn L. Culliao,1,* Rhoda S. Lumang-ay,2 Gloria Meryl T. Kingat,3 Tomasa P. Colallad,3 and Maria Victoria P. Canaria2 1Department of Medical Laboratory Science, School of Natural Science, Saint Louis University, Baguio City, Philippines 2Department of Pharmacy, School of Natural Science, Saint Louis University, Baguio City, Philippines 3Department of Biology, School of Natural Science, Saint Louis University, Baguio City, Philippines *E-mail: [email protected] ABSTRACT This study investigated the pharmacological significance of wild macrofungi collected from pine forests in Benguet and Mt. Province, Philippines, to help address a number of health-related conditions, such as oxidative stress, thrombosis, and bacterial infections. Six wild macrofungi were subjected to chloroform and ethanol extraction, and their crude extracts were screened for their total phenolic content (TPC), antioxidant, lethality, thrombolytic, and antibacterial activities. The ethanol extract of Daedaleopsis confragosa has the highest TPC at 49.28 ± 0.30 μg gallic acid equivalent (GAE)/mg extract and percent free radical inhibition activity at 74.59 ± 0.11%, which was comparable to the pure compound quercetin at 74.33 ± 0.32%. On the other hand, the ethanol extracts of Scleroderma citrinum and Postia fragilis have the most potent median effective concentration (EC50) at 431.01 ± 17.82 and 469.63 ± 15.25 μg/mL. Only the ethanol extract of Daedaleopsis confragosa exhibited low toxicity (median lethal concentration (LC50) = 565.90 μg/mL) while the rest of the test extracts are not toxic. Both chloroform and ethanol extracts of Termitomyces eurrhizus yielded the highest percent clot reduction values at 35.19 ± 0.13% and 32.41 ± 0.17%, respectively. This is the first study to report the thrombolytic activity of macrofungi extracts from the Philippines. The ethanol extract of Lenzites betulina gave the highest zone of inhibition (ZOI) against Staphylococcus aureus American Type Culture Collection (ATCC) 25923 (13.33 ± 0.58 mm) while the chloroform extract of Daedaleopsis confragosa gave the highest ZOI against Escherichia coli ATCC 25922 (12.33 ± 1.15 mm). These data indicated that the wild macrofungi tested could constitute a potential source of natural bioactive compounds in the production of pharmaceutical dosage forms or nutraceuticals. Keywords: macrofungi, total phenolic content, antioxidant, lethality, thrombolytic, antibacterial, Philippines Copyright © 2020 by De La Salle University WILD MACROFUNGI COLLECTED FROM PINE FORESTS CULLIAO ET AL. 75 INTRODUCTION (Kirk et al., 2008). Macrofungal fruiting bodies are considered as significant Bioactivity screening studies remain to nontimber forest products (Berch et al., be a fertile area for researches largely 2007), particularly as important sources of because of the ever-increasing demand for food and medicine for humans. Fungal novel, potent, and safe drugs (Griffin, 2009). specimens collected from forests have been These studies that focus on the effects of an shown to demonstrate a myriad of biological agent upon a living system are being done activities (Ajith & Janardhanan, 2007; to address human health-related concerns Wasser & Weis, 1999) and are being such as cancer, cardiovascular diseases, and consumed as nutraceuticals (Ao et al., bacterial infections, which are among the 2016). Globally, Agaricus bisporus, top leading causes of morbidity and Lentinus edodes, Pleurotus spp., and mortality worldwide (World Health Flammulina velutipes are the leading Organization, 2019). To help address these macrofungi being marketed as diseases and their associated burdens, nutraceutical foods. Ganoderma lucidum bioactivities that include antioxidant, and other Ganoderma spp., on the other lethality, thrombolytic, and antibacterial hand, have commercially available actions are being actively investigated from preparations as an anticancer agent various natural sources (Barros et al., 2008; (Valverde et al., 2015). Some Keleş et al., 2011; Obodai et al., 2014; Ogidi immunomodulating pharmaceuticals & Oyetayo, 2016; Okhuoya et al., 2010; contain β-glucans from macrofungi such as Subbulakshmi & Kannan, 2016; Toledo et ganoderan from Ganoderma lucidum, al., 2016). Aside from the high demand for grifolan from Grifola fondosa, and lentinan such agents, the continuous search for new from Lentinus edodes (Ho et al., 2020). drug sources is also being driven by the These applications all point to the potential development of adverse effects brought of macrofungi as sources of bioactive agents. about by the use of currently available However, there is a noted dearth in drugs and the emergence of bacterial current literature on natural product resistance to commonly used antibacterial researches involving Philippine macrofungi. agents, among others. Some of the recent research efforts in this Bioactive compounds that are naturally field include one that screened for the occurring in living systems have been antistaphylococcal and antioxidant widely targeted as primary sources of properties of crude ethanolic extracts from diverse molecules in several drug discovery wild macrofungi collected from Bataan studies (Liu et al., 2017; Wong et al., 2013), Province (Gaylan et al., 2018). Ragasa and in several instances, the plant or fungal (2018) reviewed and identified the chemical sources from where they are isolated have constituents of the dichloromethane been incorporated in numerous herbal extracts of commercially grown and wild supplements and health foods (Cheung, Philippine macrofungi that may contribute 2013). The search for new sources of to their anticancer activities. Aside from bioactive compounds has been directed these, ongoing researches on bioactive towards screening for the bioactivities of agents from macrofungi in the Philippines phenolic compounds naturally occurring in are rather few and limited. macrofungi (Kouassi et al., 2016). In Luzon in the Philippines, a Macrofungi are a large group of fungal continuous mountain range formed between species that produce fruiting bodies that can the southwestern areas of Mt. Province and be readily observed with the naked eye the northeastern areas of Benguet covers 76 MANILA JOURNAL OF SCIENCE VOLUME 13 (2020) some dense sections of pine forest land and was based on morphological characteristics is home to biodiverse macrofungi with as previously described (Ginns, 2017; unexplored or underexplored potential Ginns, 2019; Gonthier & Nicolotti, 2007; biological activities. Macrofungal species in Mortimer et al., 2014; Ostry et al., 2011). these areas are typically being collected for The following applicable characteristics consumption as food (Licyayo, 2018) but were utilized in morphological rarely or not at all for their biological characterization: pileus size, pileus shape, activities. This study thus aimed to screen pileus color, pileus surface features, pileus wild macrofungi for their phenolic contents margin, gill attachment, gill breadth, stipe and their potential antioxidant, lethality, location, stipe size, stipe shape, stipe color, thrombolytic, and antimicrobial activities and stipe surface features. as these may provide baseline data for the Six macrofungal species were selected production of fungal natural products or as and screened for their bioactivities, based processed pharmaceutical and on their abundance in all the collection nutraceutical products. sites, and include the following: Albatrellus ellisii, Daedaleopsis confragosa, Lenzites betulina, Postia fragilis, Scleroderma MATERIALS AND METHODS citrinum, and Termitomyces eurrhizus. Abundance of samples was operationalized Wild Macrofungi Collection and to mean a minimum of 100 g of fresh mass Identification collected from the identified sites. Other species present in the collection sites that Convenience wild macrofungi sampling were less than the cutoff mass were also from several pine forested collection sites in collected but were excluded in the the municipalities of Bauko, Mt. Province, bioactivity screening assays. and Buguias and Mankayan, Benguet Province, was conducted during the months of September and October 2019 after being Extract Preparation granted permission to do so by each of the local government units involved. The All specimens were oven dried until collection sites were at the following there are no significant changes in their positions: 16°51.1660’N, 120°51.7750’E, pre- and postdrying weights and then were 2250.8 masl; 16°51.2040’N, 120°7500’E, cut into thin sections. Fifty-gram portions of 2282.7 masl; 16°51.6690’N, 120°52.4320’E, each specimen were then exhaustively 2211.1 masl; 16°51.6730’N, 120°52.4290’E, extracted via maceration in separate 50-mL 2204.4 masl; 16°50.3740’N, 120°49.1730’E, portions of chloroform and 95% v/v ethanol 1739.8 masl; 16°50.3630’N, 120°49.1740’E, with a 48-hr interval per extraction until no 1755.3 masl; 16°50.3110’N, 120°49.1760’E, color was imparted unto the extracting 1761.3 masl; 16°51.0420’N, 120°47.2380’E, solvent. All extracts were then pooled and 1298.6 masl; and 16°51.0560’N, filtered, and the extracting solvents were 120°47.2450’E, 1321.9 masl. Whole evaporated off in a water bath maintained macrofungal specimens were taken, at 40°C (Guevara, 2004). The crude extracts wrapped in paper bags, properly labelled, were utilized as is in the
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