Toxicity, Antibacterial, and Antioxidant Activities of Fungal Endophytes Colletotrichum and Nigrospora spp. Isolated from Uvaria grandiflora
Kin Israel R. Notarte1,2*§, Mark Kevin P. Devanadera1,3§, Anna Beatriz R. Mayor1§,
Mary Christine A. Cada1, Melissa H. Pecundo1,5, and Allan Patrick G. Macabeo1,4
1The Graduate School
2Faculty of Medicine and Surgery
3Department of Biochemistry, Faculty of Pharmacy
4Laboratory for Organic Reactivity, Discovery, and Synthesis,
Research Center for the Natural and Applied Sciences (RCNAS)
University of Santo Tomas (UST), España 1015 Manila, Philippines
5South China Botanical Garden, Guangzhou, Guangdong 510650 China
§These authors contributed equally to this paper
*Corresponding Author: This email address is being protected from spambots. You need JavaScript enabled to view it.
ABSTRACT
Endophytic fungi are less explored in terms of their pharmacological applications, thus screening their phytochemical constituents and biological activities is of interest. In this study, the endophytic fungi Nigrospora and Colletotrichum spp. were isolated from the leaves of Uvaria grandiflora. The identity of the endophytes was established by molecular analysis of their fungal intergenic spacer. Biological screening showed that the fungal endophytes were most active against methicillin-resistant Staphylococcus aureus (MRSA), with the ethyl acetate broth extract of Colletotrichum sp. showing the biggest zone of inhibition (ZOI) for MRSA at 19 mm. For antibacterial activity against Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae, the ethyl acetate broth extract of Nigrospora sp. elicited better antibacterial activity (ZOI > 11 mm). Antioxidant assessment using 1,1-diphenyl-2-picrylhydrazyl (DPPH), ferric reducing antioxidant power (FRAP), and superoxide scavenging assays showed that Nigrospora broth extract had the best free radical scavenging activity with an IC50 of 3.92 mg/mL. Meanwhile, the broth extract from Colletotrichum sp. showed the best reducing power (RP50 = 4.41 mg/mL) and superoxide scavenging activity (SC50 = 0.78 mg/mL). Using Artermia salina for toxicity screening, both fungi were toxic having an LD50 < 0.40 mg/mL. The culture broth extracts showed greater antibacterial, antioxidant, and cytotoxic activities compared to the mycelial extracts. Phytochemical screening of the broth extract revealed the presence of flavonoids, sterols, phenols, and terpenoids in both fungal extracts. Tannins and coumarins were specifically detected in Colletotrichum sp., while alkaloids and indole derivatives were detected only in the Nigrospora endophyte.
INTRODUCTION
Natural products and their derivatives are clinically used to treat cancer, microbial infections, metabolic disorders, and degenerative diseases (Thomford et al. 2018). A number of these natural products are isolated from plants and microorganisms (Wright 2018). The Philippines, which is a megadiverse country, is teeming with numerous sources of natural products that remain to be explored. Among these terrestrial flora, Uvaria from the Annonaceae family is reported to possess novel phytochemicals with a wide range of pharmacological activities (Macabeo et al. 2012, 2014, 2017; Paragas et al. 2014). In particular, Uvaria grandiflora Roxb. ex Hornem. remains to be an understudied plant species with antibacterial, antioxidant, and antiprotozoal properties (Ankisetty et al. 2006, Bibak et al. 2012). Despite the potential pharmacological value of U. grandiflora, issues concerning plant biodiversity conservation remain a constraint in natural products research. Therefore, studying microbial endosymbionts within plants as alternative sources for bioactive compounds is of timely interest. These microorganisms are called endophytes that symbiotically thrive within the host plants without causing disease and can be alternative sources of bioactive chemical agents (Zhao et al. 2011). The pharmacological and industrial significance of endophytes was first demonstrated by the production of taxol (paclitaxel) by Metarhizium anisopliae, which is a fungal endosymbiont of the Taxus tree (Visalakchi and Muthumary 2010). Aside from taxol, other chemotherapeutic agents were isolated from fungal endophytes – including campthothecin, oxacillin, ampicillin, and huperzine A (Akiyama et al. 2001, Han and Rahman 2012, Nair and Padmavathy 2014). Interestingly, the endophytes associated with U. grandiflora have not yet been explored. Thus, it is the goal of our work to isolate these endophytes and screen . . . . read more
REFERENCES
AGUINALDO AM, ESPESO EI, GUEVARA BQ, NONATO MG. 2005. A Guidebook to Plant Screening: Phytochemical and Biological. Manila: University of Santo Tomas Publishing House.
AKIYAMA H, FUJII K, YAMASAKI O, OONO T, IWATSUKI K. 2001. Antibacterial action of several tannins against Staphylococcus aureus. Journal of Antimicrobial Chemotherapy 48: 487–491.
AMINIMOGHADAMFAROUJ N, NEMATOLLAH A, WIART C. 2011. Antibacterial, antioxidant activity and phytochemical study of Uvaria grandiflora: A rare species of Annonaceae. Journal of Pharmacy Research 4(4): 954–955.
ANKISETTY S, ELSOHLY HN, LI XC, KHAN SI, TEKWANI BL, SMILLIE T, WALKER L. 2006. Aromatic constituents of Uvaria grandiflora. Journal of Natural Products 66: 692–694.
ARUMUGAM GK, SRINIVASAN SK, JOSHI G, GOPAL D, RAMALINGAM K. 2014. Production and characterization of bioactive metabolites from piezotolerant deep sea fungus Nigrospora sp. in submerged fermentation. Journal of Applied Microbiology 11: 99–111.
AYOB FW, SIMARANI K, ABIDIN NZ, MOHAMAD J. 2017. First report on a novel Nigrospora sphaerica isolated from Catharanthus roseus plant with anticarcinogenic properties. Microbial Biotechnology 10: 926–932.
BALESDENT MH, JEDRYCZKA M, JAIN L, MENDES-PEREIRA E, BERTRANDY J, ROUXEL T. 1998. Conidia as a substrate for internal transcribed spacer‐based PCR identification of members of the Leptosphaeria maculans species complex. Phytopathology 88: 1210–1217.
BIBAK B, KHAKSHOR A, KAMALI H, AHMADZADEH GHAVIDEL R, AMINIMOGHADAMFAROUJ N. 2012. Evaluation of antibacterial properties, phytochemical contents and antioxidant capacities of leaf and stem barks of Uvaria grandiflora Roxb. Journal of North Khorasan University of Medical Sciences 4: 113–119.
DOS SANTOS IP, DA SILVA LCN, DA SILVA MV, DE ARAUJO JM, CAVALCANTI MS, LIMA VLM. 2015. Antibacterial activity of endophytic fungi from leaves of Indigofera suffruticosa Miller (Fabaceae). Frontiers in Microbiology 6: 1–7.
GUNATILAKA AA. 2006. Natural products from plant-associated microorganisms: Distribution, structural diversity, bioactivity, and implications of their occurrence. Journal of Natural Products 69(3): 509–526.
HAN T, RAHMAN K. 2012. Alkaloids produced by endophytic fungi: A review. Natural Product Communications 7: 963–968.
MACABEO APG, LETADA AG, BUDDE S, FADERL C, DAHSE HM, FRANZBLAU SG, ALEJANDRO GJD, PIERENS GK, GARSON MJ. 2017. Antitubercular and cytotoxic chlorinated seco-cyclohexenes from Uvaria alba. Journal of Natural Products 80(12): 3319–3323.
MACABEO APG, MARTINEZ FPA, KURTAN T, TOTH L, MANDI A, SCHMIDT S, HEILMANN J, ALEJANDRO GJ, KNORN M, DAHSE HM, FRANZBLAU, SG. 2014. Tetrahydroxanthene-1,3(2H)-dione derivatives from Uvaria valderramensis. Journal of Natural Products 77(12): 2711–2715.
MACABEO APG, TUDLA FA, KHRON K, FRANZBLAU SG. 2012. Antitubercular activity of the semi-polar extractives of Uvaria rufa. Asian Pacific Journal of Tropical Medicine 5:777–780.
MEYER B, FERRIGHI N, PUTNAM J, JACOBSEN L, NICHOLS D, MCLAUGHLIN J. 1982. Brine shrimp: A convenient general bioassay for active plant constituents. Planta Medica 45: 31–34.
NAIR DN, PADMAVATHY S. 2014. Impact of endophytic microorganisms on plants, environment and humans. The Scientific World Journal 2014: 1–11.
QUIMIO TH. 1988. Illustrated Philippine fungi. Manila: National Bookstore.
QUINTO E, SANTOS M. 2005. A Guidebook to Plant Screening: Phytochemical and Biological. Manila: University of Santo Tomas Publishing House.
PACKIARAJ R, JEYAKUMAR S, AYYAPPAN N, ADHIRAJAN N, PREMKUMAR G, RAJARATHINAM K, MUTHURAMKUMAR S. 2016. Antimicrobial and cytotoxic activities of endophytic fungus Colletotrichum gloeosporioides isolated from endemic tree Cinnamomum malabatrum. Studies in Fungi 1: 104–113
PARAGAS E, DIETMAR G, KROHN K, FRANZBLAU SG, MACABEO APG. 2014. Antitubercular flavonol derivatives from Uvaria rufa. Research Journal of Pharmaceutical, Biological and Chemical Sciences 5: 856–859.
PATIL RH, PATIL MP, MAHESHWARI VL. 2016. Studies in Natural Products Chemistry. UK: Elsevier.
PAWLE G, SINGH SK. 2014. Antimicrobial, antioxidant activity and phytochemical analysis of an endophytic species of Nigrospora isolated from living fossil Ginkgo biloba. Current Research in Environmental and Applied Mycology 4(1): 1–9.
RAPER KB, FENNELL DI. 1977. The genus Aspergillus. New York: Robert E. Krieger Publishing Co.
ROSSETO P, COSTA AT, POLONIO JC, SILVA AA, PAMPHILE JA, AZEVEDO JL. 2016. Investigation of mycoviruses in endophytic and phytopathogenic strains of Colletotrichum from different hosts. Genetics and Molecular Research 15(1): 1–11.
SANTIAGO LA, VALERIO VLM. 2013. Assessment of antioxidant activities of crude ethanolic leaf extract of Ficus pseudopalma Blanco (Moraceae). International Journal of Pharmaceutical Research 3(1): 15–25.
SCHULZ B, WANKE U, DRAEGER S, AUST HJ. 1993. Endophytes from herbaceous plants and shrubs: Effectiveness of surface sterilization methods. Mycological Research 97: 1447–1450.
TIANPANICH K, PRACHYA S, WIYAKRUTTA S, MAHIDOL C, RUCHIRAWAT S, KITTAKOOP P. 2011. Radical scavenging and antioxidant activities of isocoumarins and a phthalide from the endophytic fungus Colletotrichum sp. Journal of Natural Products 74: 79–81.
THOMFORD NE, SENTHEBANE DA, ROWE A, MUNRO D, SEELE P, MAROYI A, DZOBO K. 2018. Natural products for drug discovery in the 21st century: Innovations for novel drug discovery. International Journal of Molecular Sciences 19(6): 1–29.
THRANE U, ANDERSEN B, FRISVAD JC, SMEDSGAARD J. 2007. Metabolomics: A powerful tool in systems biology. Berlin: Springer.
VASUNDHARA M, KUMAR A, REDDY MS. 2016. Molecular approaches to screen bioactive compounds from endophytic fungi. Frontiers in Microbiology 7(1774): 1–12.
VERMA VC, KHARWAR RN, STROBEL G. 2009. Chemical and functional diversity of natural products from plant-associated endophytic fungi. Natural Product Communications 4: 1511–1532.
VISALAKCHI S, MUTHUMARY J. 2010. Taxol (anticancer drug) producing endophytic fungi: An overview. International Journal of Pharmacy and Biological Sciences 1: 1–9.
WRIGHT GD. 2018. Unlocking the potential of natural products in drug discovery. Microbial Biotechnology 12(1): 55–57.
WU Z, XIE Z, WU M, LI X, LI W, DING W, SHE Z, LI C. 2018. New antimicrobial cyclopentenones from Nigrospora sphaerica ZMT05, a fungus derived from Oxya chinensis Thunber. Journal of Agricultural and Food Chemistry 66: 5368–5372.
ZHAO J, SHAN T, MOU Y, ZHOU L. 2011. Plant-derived bioactive compounds produced by endophytic fungi. Mini-Reviews in Medicinal Chemistry 11(2): 159.
