Antimicrobial and antioxidant activities of endophytic fungi extracts isolated from Carissa carandas

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.


INTRODUCTION
Nowadays, drug resistant microorganisms and free radical agents are gaining more attention.They lead to infectious diseases in humans and to various other diseases (cancer, asthma and cardiovascular disease).Infectious disease and free radicals are important issues found in all regions of the world (Racek et al., 2001;Hubalek, 2003;Lobo et al., 2010;Lindahl and Grace, 2015).Increasing attention has been paid to natural products, especially those from fungal endophytes.It is well documented that fungal endophytes are a good source of bioactive natural compounds that are effective, have low toxicity, and cause a minimum environmental impact (Jalgaonwala et al., 2011;Nisa et al., 2015).There are several definitions of endophytic fungi; one of the recent definitions is that it is fungus which colonizes host plant tissue without visible symptoms (Jia et al., 2016;Gouda et al., 2016).Recent studies have reported that the functional potential of fungal endophytes is derived from many terrestrial plant species (Joseph and Priya, 2011).In some cases, novel compounds from fungal *Corresponding author.E-mail: preuttiporn.suppaphon@gmail.com.
Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution Li cense 4.0 International License endophytes showed strong antimicrobial activities against pathogenic microorganisms such as antibacterial, antifungal and anti-parasitic, and had strong antioxidant activities (Ascencio et al., 2014;Brissow et al., 2017;Raunsai et al., 2018).Active compounds (trichodermin and volatile compound) isolated from the fungal endophyte Trichoderma species had a strong activity against pathogenic bacteria and pathogenic fungi (Leylaie and Zafari, 2018).Fusaripeptide A isolated from the fungal endophyte Fusarium species which is isolated from roots of Mentha longifolia L. showed strong activity against Plasmodium falciparum (antimalarial) with an IC 50 value of 0.34 µM, and against Candida albicans, Candida glabrata, Candida krusei and Aspergillus fumigates (antifungal) with IC 50 values of 0.11, 0.24, 0.19 and 0.14 µM, respectively (Ibrahim et al., 2015).Furthermore, fungal endophytes can produce active antioxidant compounds (Huang et al., 2007;Khiralla et al., 2015).Bioactive natural products from fungal endophytes, in particular from Carissa carandas, have been rarely studied (Yadav et al., 2014;Tenguria andFirodiya, 2015, 2016).Previous studies on fungal endophytes which were isolated from C. carandas have been focused on biodiversity, distribution and cytotoxicity of endophytic extracts (Tenguria et al., 2012;Tenguria and Firodiya, 2015).
Carissa is a medicinal plant belonging to the Apocynaceae family comprised 20 to 30 species and is found in many parts of Asia, Africa and Australia.The common species of this genus are C. carandas, Carissa macrocarpa, Carissa grandiflora, Carissa edulis, Carissa spinarum, Carissa lanceolata, Carissa opaca, Carissa congesta and Carissa bispinosa.Many parts of this plant (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 dysfunction, fever, digestion system problems and diarrheas (Arif et al., 2016).Recently, Toobpeng et al. (2017) reported the antibacterial activity of fruit extracts against Pseudomonas aeruginosa, Klebsiella pneumonia, Escherichia coli, Staphyloccus aureus, methicillinresistant S. aureus, Acinetobacter baumannii, Enterococcus faecalis, and Schleichera oleosa.However, the biological activities, especially, antimicrobial and antioxidant activities of fungal endophytes from C. carandas have not been studied.
Thus, this study aimed to isolate endophytic fungi from healthy fresh leaves of C. carandas and to screen these microorganisms for their ability to produce antimicrobial metabolites against human pathogens and antioxidant substances that inhibit or delay the oxidation of biologically relevant molecules.

Sample collection and fungal isolation
Healthy leaf samples of C. carandas were randomly collected Supaphon and Preedanon 465 during January to March 2018 from Trang,Thailand (7° 33' 22.79" N;99° 36' 41.08" E).Samples were surface-sterilized with 10% ethanol (5 min), 3% sodium hypochlorite (15 s), and 10% ethanol (5 min), and rinsed with distilled water and dried on sterile tissue paper.Each of the leaf parts was cut into five segments (0.5 cm 2 ) and put onto Potato Dextrose Agar (PDA) provided with antibiotics (50 mg/L penicillin and streptomycin).Plates were incubated at room temperature for 4 weeks.Fungal isolates were subcultured in PDA without antibiotics until they were pure cultures.All fungal isolates were identified and selected for further study based on their morphological characteristics after the incubation period.Each pure fungal isolate on PDA was cut into small pieces and maintained in 20% glycerol at -80°C.

Fermentations and extractions
Fermentation and extraction methods were conducted according to Supaphon et al. (2010Supaphon et al. ( , 2013) ) with some modifications.Six agar plugs (1 cm 2 ) of fungal mycelium were inoculated in 500 mL Erlenmeyer flasks containing 250 mL potato dextrose broth (PDB) and were incubated for 3 weeks at room temperature for production of metabolites.The culture broth was filtered to separate the filtrate and mycelia.The filtrate was extracted with an equal volume of ethyl acetate (EtOAc) in a separating funnel two times.The combined EtOAc extracts were evaporated to dryness under reduced pressure at 45°C using a rotary vacuum evaporator to obtain the broth ethyl acetate extract (BE extract).The fungal mycelia were soaked in 500 mL of methanol (MeOH) for 3 days.The aqueous MeOH layer was concentrated using rotary evaporator to give the aqueous layer.The aqueous layer was extracted with an equal volume of hexane two times, followed by EtOAc twice.The combined EtOAc and hexane extracts were evaporated to dryness under reduced pressure at 45°C using a rotary vacuum evaporator to obtain the cell ethyl acetate and cell hexane extracts (CE and CH extracts).The stock extracts were kept in vials at 4°C.Working extracts were kept in vials at room temperature until used.

Antimicrobial assay
The dried extracts were dissolved in dimethyl sulfoxide (DMSO) to prepare stock solutions at 100 mg/mL.All the extracts at a final concentration of 200 µg/mL were screened for their antimicrobial activity against ten pathogenic microorganisms, including  CLSI 2008CLSI , 2012)).Microtiter plates were incubated at 35°C for 15 h, then 30 µL of resazurin solution (0.18%) was added to each well and incubated for 3 h under the same conditions (Supaphon et al., 2018).After incubation, the results were recorded as positive (blue color indicated growth inhibition) and negative (pink color indicated microbial growth).After that, the active extracts from the screening 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 of extract that inhibited growth was recorded as MIC.The concentration of extract at MIC and more than MIC were determined by streaking onto nutrient agar (NA) plates for bacteria, Sabouraud's dextrose agar (SDA) plates for yeasts and PDA plates for filamentous fungi and incubated under appropriate conditions.The lowest concentration of extract that showed no growth was recorded as the minimum bactericidal concentration (MBC) for yeast and the minimum fungicidal concentration (MFC) for filamentous fungi.Commercial antibiotics were used as standard agents for positive inhibitory controls (vancomycin for Gram-positive bacteria, gentamicin for Gramnegative bacteria, amphotericin for yeasts and T. marneffei and miconazole for M. gypseum).

Scanning electron microscopy analysis
The effect of the most active extract on cell morphology was determined using scanning electron microscopy (SEM).The sample was prepared according to previous studies with slight modifications (Supaphon et al., 2018).Briefly, cell suspension (10 8 CFU/mL) was treated with four times MIC concentration of the active extract and incubated for 24 h.For the controls, antibiotics and DMSO were used as positive and negative controls, respectively.The treatments were fixed with 2.5% glutaraldehyde in a phosphate buffer solution for 1 h and washed three times with PBS, pH 7.2.Each treatment was serially dehydrated with 50, 70, 80, 90 and 100% ethanol.Then, cell samples were dried in a lyophilizer, smeared on a silver stub, mounted with gold and observed using SEM at the Scientific Equipment Center, Prince of Songkla University.

Gas chromatography-mass spectrometry analysis (GC-MS)
GC-MS was used to determine the active extracts.This experiment was performed according to the previous study with some modifications (Supaphon et al., 2018).The separation and identification of the compounds (extract from Nigrospora guilinensis) used the HP5MS capillary column (30 m  0.25 mm 0.5 µm).The temperature program was performed as follows: initial temperature of 50°C (2 min), raised to 160°C at the rate of 8°C/min (5 min), then raised to 270°C at the rate of 8°C/min (8 min), Helium was used as the carrier gas at the rate of 10 mL/min.The fragmentation of the MS range from 40 to 1000 m/z was conducted by electronic impact mode (ionization energy, 70 eV, 300°C) and scanned at the rate of 3.0 scans/s.GC-MS was analyzed for 50 min.

Determination of antioxidant activity (DPPH) assay
1, 1-diphenyl-2-picryl-hydrazyl (DPPH) scavenging activity of endophytic fungi extracts was evaluated according to the previous report of Yadav et al. (2014) with slight modifications.Briefly, the DPPH solution was prepared by dissolving 2.4 mg DPPH in 100 mL methanol, and the stock solution was kept at -20°C until used.The extract solution at a concentration of 10 mg/mL (50 μL) was added to 50 μL of 1 mM DPPH solution in 96-well microtiter plates.The mixture was shaken and stored at room temperature for 30 min in the dark, and then the absorbance was recorded at 515 nm using a spectrophotometer.For the control, the absorbance of ascorbic acid solution was used as a standard for making the calibration curve using 0.2, 0.4, 0.6, 0.8, and 1.0 mg/mL concentrations of the standard.All determinations were performed in triplicate.The percent scavenging activity was calculated using the formula: [A0-As/A0] × 100, where A0 and As represent the absorbance values of the control and extract, respectively.The active extracts that provided ≥50% scavenging activity were identified by interpolation from linear regression analysis.

Determination of total phenolics contents
The total phenolic content of the broth ethyl acetate extracts was measured as described by Yadav et al. ( 2014) with some modification.The extracts were prepared and diluted from stock solutions (1 mg/mL).100 µL of fungi extract was diluted with 7 mL water and then was mixed with 500 µL of Folin-Ciocalte.The mixture solution was incubated at room temperature for 4 min.Then 1.5 mL of sodium carbonate (7.5%) was added and kept in the dark at room temperature for 2 h.The results were recorded by measuring the absorbance at 765 nm using a spectrophotometer (Thermo Scientific).Gallic acid was used as positive control, and a standard curve at the concentrations of 0.2, 0.4, 0.6, 0.8 and 1 mg/mL in 70% of methanol was constructed.The results were reported as mg gallic acid equivalent dry weight (GAE/g of extract).
Each treatment was performed in triplicate.

Molecular traits
The most active isolate as determined by the antimicrobial and antioxidant activity assays was further identified by molecular methods based on ITS sequence analyses.Extraction of fungal mycelium used the modified CTAB method from O'Donnell et al. (1997).Afterwards, the qualities of genomic DNA were estimated with 1% agarose gel electrophoresis in 1% TAE buffer.Internal transcribed spacer (ITS) rDNA was amplified by PCR with the primer pairs ITS5/ITS4 (White et al., 1990).The PCR reactions used Taq DNA polymerase (Thermo Scientific) following the manufacturer's instructions and were performed in a T100TM Thermal cycler (BIO-RAD laboratories, Inc).Furthermore, the amplification conditions of ITS rDNA were performed as follows: initial denaturation at 94°C for 2 min, followed by 35 cycles at 94°C for 1 min, annealing at 55°C for 1 min and elongation at 72°C for 2 min, with a final extension period of 72°C for 10 min.The PCR products were checked on 1% agarose electrophoresis gels and stained with RedSafe DNA stain (20,000X).After that, PCR products were purified by Macrogen Inc. in South Korea for direct DNA sequencing.

Phylogenetic analysis
Nucleotide sequences in this study were compared with the related sequences from GenBank (http://www.ncbinlm.nih.gov) and following Wang et al. (2017).All sequences were assembled with BioEdit version 7.2.5 (Hall, 2005) and aligned with Muscle program version 3.8 (Edgar, 2004).While, the phylogenetic tree was constructed by maximum parsimony (MP) and maximum likelihood (ML) analyses.Maximum parsimony analyses were performed in PAUP*4.0b10(Swofford, 2002).The most parsimonious trees (MPTs) results were evaluated, followed by heuristic searches: 100 replicates of random stepwise addition of sequence, branchswapping algorithm: tree-bisection-reconnection (TBR) and equal weight characters.Maximum parsimony bootstrap supports of the clades were approximated by 1000 replicates (stepwise addition of sequence, 10 replicates of random addition of taxa, TBR branchingswapping algorithm).Additionally, the maximum likelihood and bootstrap analyses were generated on the CIPRES web portal (Miller et al., 2010) through RAxML 8.2.4 (Stamatakis, 2014) with the BFGS method to optimize GTR rate parameters.The phylograms were visualized using FigTree v1.4.3 (Rambaut, 2016).Moreover, the sequences analyzed in this study were deposited in the GenBank databases and are shown in Table 4.The alignment result was submitted to TreeBASE (submission number: 24397).

Fungal isolation
One-hundred and nine fungal isolates from 900 segments of C. carandas were identified based on their morphology as Penicillium, 20 isolates; Cladosporium, 13 isolates; Fusarium, 6 isolates; Curvularia, 5 isolates; and Nigrospora 3 isolates.The remaining fungal isolates (62 isolates) did not produce any reproductive structure and were classified as unidentified endophytic fungi.All endophytic fungal isolates were grouped into 36 morphotypes based on their morphology.Representative isolates were selected from each group for antimicrobial and antioxidant activity tests.

Antimicrobial assay
The preliminary screening of crude extracts (108 extracts) from selected endophytic fungi (36 isolates) revealed the presence of bioactive compounds.The antimicrobial activity was determined by using the colorimetric broth microdilution assay.1.

The SEM analysis
The cell surface morphology of C. neoformans ATCC90112 was observed after treatment with the most active extract (BE extract from TSU-EFHA009) using SEM.Cells after treatment with the extract at a concentration of 4X MIC (16 µg/mL) appeared to be shrunken, with broken and wrinkled cell surfaces, similar to cells treated with amphotericin B at a concentration of 1 µg/mL (positive control).Whereas, the morphology of cells treated with 1% DMSO (negative control) exhibited a normal cell surface without any damage (Figure 1).These SEM analyses indicated that the BE extract had effects on cell wall of C. neoformans.

Antioxidant activity and total phenolic content
Crude extracts of 36 fungal isolates were screened for Supaphon and Preedanon 467 their antioxidant properties (DPPH assay) in comparison with an antioxidant agent (ascorbic acid).From the DPPH radical scavenging activity results, three out of the 108 extracts showed strong antioxidant activity with 90% inhibition, while ascorbic acid gave a 95% inhibition.Among the active extracts (three extracts) had an excellent scavenging effect, especially the BE from TSU-EFHA009.The inhibitory concentration value (IC 50 ) of this extract was 0.03 mg/mL (Table 2).

Phylogenetic relationship of active fungal isolates
ITS rDNA sequences analyses were used to classify the selected endophytic fungi.The phylogenetic trees were performed by MP and ML analyses.The sequence similarity of sequences retrieved from GenBank databases was determined.Subsequently, BLAST search results of ITS rDNA sequences indicated that the isolates belonged to the class Sordariomycetes, order Xylariales, and family Apiosporaceae.The generated phylogenetic alignment consisted of 48 taxa (Table 4), with Amphisphaeria sorbi (MFLUCC 13-0721), Phlogicylindrium eucalyptorum (CBS111689) and Phlogicylindrium uniforme (CBS131312) as outgroup.
The dataset constituted 607 total characters; 385 characters were constant; 191 characters were parsimony informative and 31 variable characters were parsimony uninformative.The best tree inferred a length of 443 steps [consistency index (CI) = 0.693, retention index (RI) = 0.873, relative consistency index (RC) = 0.605, homoplasy index (HI) = 0.307].One of the ten MPTs is as shown in Figeru 2; the best topology was determined by the K-H test (Kishino and Hasegawa, 1989).The maximum likelihood tree illustrated a similar topology to the MP tree (data not shown).
The phylogenetic results demonstrated that our strain (TSU-EFHA009) was assigned to genus Nigrospora with strong statistic support as shown in Figure 2. It is grouped together with the N. guilinensis clade (LC1843 and LC7301), with strong support (65% BSMP and 64% BSML) and their numbers of nucleotide substitutions exhibited 507/515 = 98.5% similarity with eight substitutions.Hence, this strain should be classified taxonomically as N. guilinensis.

GC-MS analysis
The CE extract from isolate TSU-EFHA 009 was analyzed by GC-MS as shown in Table 3.In this study, retention indices were also compared to the published values.An agreement above 90%, of the spectra, was considered for identification of constituents.The CE could be divided into 10 components, the majority of which are 4-(cyclopentyloxy) cyclohex-2-en-1-yl acetate (21.89%), 5,7a-dimethyloctahydro-1-inden-3a-yl)(phenyl)methanone (12.37%) and 2-methylcyclohexanone (10.02%).Three main components were identified in the BE fraction and represented over 10% of the peak area.The remaining components were present at ˂10% of the peak area.Many secondary metabolites are produced by the fungal endophytes.Thus, this study aimed to evaluate the antimicrobial and antioxidant activities of such fungal isolates.They may be a renewable source of novel bactericidal, fungicidal and antioxidant activities.The effects of the endophytic extracts in this study against tested pathogenic microorganisms were significant, except against Gram-negative bacteria.This might be because of the structure of these bacteria.There is an outer membrane that prevents a sufficient active agent   (Beveridge, 1999).This is the first report that evaluated the antimicrobial and antioxidant properties of endophytic fungi from C. carandas, while other reports focused on the biological activities and compound identification from C. carandas.Pawle and Singh (2014) isolated fungal endophytes (Nigrospora species) from the living fossil Ginkgo biloba.The ethyl acetate extracts from culture broth showed antimicrobial activity with MIC values of 2.5 mg/mL against E. coli, Klebsiella species, S. aureus, C. albicans and Geotrichum species, while the ethyl acetate extract from N. guilinensis TSU-EFHA009 in this study yielded high activity against C. neoformans with an MIC value of 4 µg/mL and MFC of 8 µg/mL.These results give credence that extracts from fungal endophytes show great antimicrobial activity.Molecular identification showed that the active fungi could be classified as Sordariomycetes and identified as N. guilinensis; which was also reported to be the major endophytic group from various plant species (Zhang and Yao, 2015).Nigrospora spp. was commonly found as an endophyte in several species of plants (Sharma and Rangari, 2015;Tenguria and Firodiya, 2015;Kucerova-Chlupacova et al., 2016;Saad et al., 2019).Furthermore, there are many reports about metabolites from Nigrospora spp.(Arumugam et al., 2014;Rathod et al., 2014;Ibrahim et al., 2018) that displayed good activity against pathogenic microorganisms.Some secondary metabolites (griseofulvin, spirobenzofuran and pyrazine) from Nigrospora spp.have been reported as antifungal and antibacterial substances (Kratky et al., 2012;Roymahapatra et al., 2012;Sharma and Rangari, 2015;Kucerova-Chlupacova et al., 2016).In addition, the ethyl acetate extract of N. guilinensis (TSU-EFHA009) contained three main compounds which are 4-(cyclopentyloxy)cyclohex-2-en-1yl acetate, 5,7a-dimethyloctahydro-1-inden-3a-yl)(phenyl) methanone and 2-methylcyclohexanone.It is possible that they might have an important role in antimicrobial and antioxidant activities.However, these compounds have not been previously reported from Nigrospora spp.and there are no reports about their biological activities.
Phenolic compounds are secondary metabolites that stabilize lipid oxidation.The amount of phenolic content in crude extracts seems to have an important role in antioxidant activity.The total phenolic content of fungal extracts has been previously determined (Bharwaj et al., 2015;Madhuchhanda et al., 2017).Total phenolic content in this study ranged from 4.30±0.25 to 41.20±0.40gallic acid equivalents (GAE mg/g of extract) of dry weight of extracts (Table 2).Total phenolic content was lower than the extracts of endophytic fungi from Eugenia jambolana.The endophytic extracts having high phenolic contents showed a high antioxidant activity which ranged from 58 to 60 GAE mg/g of extract and produced a 50 to 80% inhibition (Yadav et al., 2014).The antioxidant content range in this study was different from previous studies   and this may be due to the fungal strains and extraction method (Srinivansan et al., 2010;Chowdhury et al., 2018).However, the results in this study confirmed that the endophytic fungus (TSU-EFHA009) has a high phenolic content and showed excellent activity against DPPH radicals.

Conclusions
Broth ethyl acetate extract of endophytic fungus N. guilinensis TSU-EFHA009 significantly showed strong antimicrobial and antioxidant activities.This finding confirms that endophytic fungi isolated from C. carandas were sources of the potential substances.Thus, this plant appears to be an interesting plant which harbors active fungal isolates for development as pharmaceutical agents in the future.

Figure 2 .
Figure 2. The most parsimonious trees using the ITS rDNA gene with Amphisphaeria sorbi (MFLUCC 13-0721), Phlogicylindrium eucalyptorum (CBS111689) and P. uniforme (CBS131312) as outgroup.The best phylogeny performed by maximum parsimony analyses.Maximum parsimony bootstrap values (BSMP, left) and maximum likelihood (BSML, right) bootstrap values equal or greater than 50% are shown above each branch.The bold line shows strong supported by all bootstrap values (100%).

Table 2 .
DPPH scavenging activity and total phenolic content of the potential crude extracts.

Table 3 .
Major constituents of broth ethyl acetate (BE) fraction of N. guilinensis.

Table 4 .
GenBank accession number of generated ITS rDNA sequences.