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Antimicrobial and Antioxidant Activities of Endophytic Fungi Extracts Isolated from Carissa Carandas

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Antimicrobial and Antioxidant Activities of Endophytic Fungi Extracts Isolated from Carissa Carandas Vol. 13(27), pp. 464-473, September, 2019 DOI: 10.5897/AJMR2019.9164 Article Number: 2CC499E61808 ISSN: 1996-0808 Copyright ©2019 Author(s) retain the copyright of this article African Journal of Microbiology Research http://www.academicjournals.org/AJMR Full Length Research Paper Antimicrobial and antioxidant activities of endophytic fungi extracts isolated from Carissa carandas Preuttiporn Supaphon1* and Sita Preedanon2 1Department of Biology, Faculty of Science, Thaksin University, Papayom, Phatthalung, 93210 Thailand. 2National Center for Genetic Engineering and Biotechnology (BIOTEC), Thailand Science Park, Phaholyothin Road, Klong 1, Klong Luang, Pathumthani, 12120 Thailand. Received 2 July, 2019; Accepted 22 August, 2019. This is the first report of endophytic fungi derived from Carissa carandas producing bioactive compounds in Thailand. The aims of this research were to evaluate the antimicrobial and antioxidant activities of extracts from endophytic fungi, identify the potential fungal isolates by phylogenetic analysis and analyze the composition of the potential crude extract by gas chromatography-mass spectrometry (GC-MS). The endophytic fungus Nigrospora guilinensis TSU-EFHA009 produced the most active extracts. Broth ethyl acetate extract (BE) had the strongest activity against Cryptococcus neoformans at a minimum inhibitory concentration (MIC) of 4 µg/mL and a minimum fungicidal concentration of 8 µg/mL. Moreover, the antimicrobial activity was confirmed using scanning electron microscopy. The target cells were morphologically damaged. In addition, this active extract had the highest antioxidant activity with an inhibitory concentration (IC50) value of 0.03 mg/mL. The total phenolic content of the target extract was detected by using the colorimetric method. This extract contained a total phenolic content of 41.20±0.40 mg GAE/g of the extract. The results indicated that the endophytic fungi from C. carandas are good sources of antimicrobial and antioxidant substances. Key words: Antimicrobial activity, antioxidant activity, endophytic fungi, active metabolites. INTRODUCTION Nowadays, drug resistant microorganisms and free source of bioactive natural compounds that are effective, radical agents are gaining more attention. They lead to have low toxicity, and cause a minimum environmental infectious diseases in humans and to various other impact (Jalgaonwala et al., 2011; Nisa et al., 2015). There diseases (cancer, asthma and cardiovascular disease). are several definitions of endophytic fungi; one of the Infectious disease and free radicals are important issues recent definitions is that it is fungus which colonizes host found in all regions of the world (Racek et al., 2001; plant tissue without visible symptoms (Jia et al., 2016; Hubalek, 2003; Lobo et al., 2010; Lindahl and Grace, Gouda et al., 2016). Recent studies have reported that 2015). Increasing attention has been paid to natural the functional potential of fungal endophytes is derived products, especially those from fungal endophytes. It is from many terrestrial plant species (Joseph and Priya, well documented that fungal endophytes are a good 2011). In some cases, novel compounds from fungal *Corresponding author. E-mail: [email protected]. Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution Li cense 4.0 International License Supaphon and Preedanon 465 endophytes showed strong antimicrobial activities against during January to March 2018 from Trang, Thailand (7° 33' 22.79" pathogenic microorganisms such as antibacterial, N; 99° 36' 41.08" E). Samples were surface-sterilized with 10% antifungal and anti-parasitic, and had strong antioxidant ethanol (5 min), 3% sodium hypochlorite (15 s), and 10% ethanol (5 min), and rinsed with distilled water and dried on sterile tissue paper. activities (Ascencio et al., 2014; Brissow et al., 2017; Each of the leaf parts was cut into five segments (0.5 cm2) and put Raunsai et al., 2018). Active compounds (trichodermin onto Potato Dextrose Agar (PDA) provided with antibiotics (50 mg/L and volatile compound) isolated from the fungal penicillin and streptomycin). Plates were incubated at room endophyte Trichoderma species had a strong activity temperature for 4 weeks. Fungal isolates were subcultured in PDA against pathogenic bacteria and pathogenic fungi (Leylaie without antibiotics until they were pure cultures. All fungal isolates were identified and selected for further study based on their and Zafari, 2018). Fusaripeptide A isolated from the morphological characteristics after the incubation period. Each pure fungal endophyte Fusarium species which is isolated fungal isolate on PDA was cut into small pieces and maintained in from roots of Mentha longifolia L. showed strong activity 20% glycerol at -80°C. against Plasmodium falciparum (antimalarial) with an IC50 value of 0.34 µM, and against Candida albicans, Candida glabrata, Candida krusei and Aspergillus fumigates Fermentations and extractions (antifungal) with IC50 values of 0.11, 0.24, 0.19 and 0.14 Fermentation and extraction methods were conducted according to µM, respectively (Ibrahim et al., 2015). Furthermore, Supaphon et al. (2010, 2013) with some modifications. Six agar fungal endophytes can produce active antioxidant plugs (1 cm2) of fungal mycelium were inoculated in 500 mL compounds (Huang et al., 2007; Khiralla et al., 2015). Erlenmeyer flasks containing 250 mL potato dextrose broth (PDB) Bioactive natural products from fungal endophytes, in and were incubated for 3 weeks at room temperature for production of metabolites. The culture broth was filtered to separate the filtrate particular from Carissa carandas, have been rarely studied and mycelia. The filtrate was extracted with an equal volume of (Yadav et al., 2014; Tenguria and Firodiya, 2015, 2016). ethyl acetate (EtOAc) in a separating funnel two times. The Previous studies on fungal endophytes which were isolated combined EtOAc extracts were evaporated to dryness under from C. carandas have been focused on biodiversity, reduced pressure at 45°C using a rotary vacuum evaporator to distribution and cytotoxicity of endophytic extracts obtain the broth ethyl acetate extract (BE extract). The fungal (Tenguria et al., 2012; Tenguria and Firodiya, 2015). mycelia were soaked in 500 mL of methanol (MeOH) for 3 days. The aqueous MeOH layer was concentrated using rotary Carissa is a medicinal plant belonging to the evaporator to give the aqueous layer. The aqueous layer was Apocynaceae family comprised 20 to 30 species and is extracted with an equal volume of hexane two times, followed by found in many parts of Asia, Africa and Australia. The EtOAc twice. The combined EtOAc and hexane extracts were common species of this genus are C. carandas, Carissa evaporated to dryness under reduced pressure at 45°C using a macrocarpa, Carissa grandiflora, Carissa edulis, Carissa rotary vacuum evaporator to obtain the cell ethyl acetate and cell spinarum, Carissa lanceolata, Carissa opaca, Carissa hexane extracts (CE and CH extracts). The stock extracts were kept in vials at 4°C. Working extracts were kept in vials at room congesta and Carissa bispinosa. Many parts of this plant temperature until used. (stem, root, bark, fruit and seed) have been used in traditional medicine for thousands of years. Its fruit are used as a treatment for various ailments such as liver Antimicrobial assay dysfunction, fever, digestion system problems and The dried extracts were dissolved in dimethyl sulfoxide (DMSO) to diarrheas (Arif et al., 2016). Recently, Toobpeng et al. prepare stock solutions at 100 mg/mL. All the extracts at a final (2017) reported the antibacterial activity of fruit extracts concentration of 200 µg/mL were screened for their antimicrobial against Pseudomonas aeruginosa, Klebsiella pneumonia, activity against ten pathogenic microorganisms, including S. aureus Escherichia coli, Staphyloccus aureus, methicillin- ATCC25923, a clinical isolate of methicillin-resistant S. aureus resistant S. aureus, Acinetobacter baumannii, (MRSA) SK1, E. coli ATCC25922, and P. aeruginosa ATCC27853, C. albicans ATCC90028, C. albicans NCPF3153, Cryptococcus Enterococcus faecalis, and Schleichera oleosa. However, neoformans ATCC90112, C. neoformans ATCC90113, a clinical the biological activities, especially, antimicrobial and isolate of Microsporum gypseum and Talaromyces marneffei by the antioxidant activities of fungal endophytes from C. colorimetric microdilution method according to the Clinical and carandas have not been studied. Laboratory Standards Institute (CLSI) with slight modifications Thus, this study aimed to isolate endophytic fungi from (CLSI 2008, 2012). Microtiter plates were incubated at 35°C for 15 healthy fresh leaves of C. carandas and to screen these h, then 30 µL of resazurin solution (0.18%) was added to each well microorganisms for their ability to produce antimicrobial and incubated for 3 h under the same conditions (Supaphon et al., 2018). After incubation, the results were recorded as positive (blue metabolites against human pathogens and antioxidant color indicated growth inhibition) and negative (pink color indicated substances that inhibit or delay the oxidation of microbial growth). After that, the active extracts from the screening biologically relevant molecules. test were determined by the same method for the minimum inhibitory concentrations (MICs) at a concentration range of 0.25 to 128 µg/mL. The lowest concentration
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