Extracts of endophytic fungi from leaves of selected Nigerian ethnomedicinal plants exhibited antioxidant activity

Background Plants with an ethnobotanical history are known to harbor diverse group of endophytic fungi, which constitute major natural sources of bioactive compounds. In the present study, we evaluated the antioxidant activity of endophytic fungi from eight Nigerian ethnomedicinal plants. Endophytic fungi were isolated from the leaves of Acalypha ornata, Albizia zygia, Alchornea cordifolia, Chrysophyllum albidum, Ficus exasperata, Gomphrena celosioides, Millettia thonningii, and Newbouldia laevis. Methods Endophytic fungi were isolated from the leaves of selected plants via surface sterilization. Isolated fungi were identified by internal transcribed spacer (ITS-rDNA) sequence analysis. Pure fungal strains were subjected to fermentation process on solid rice medium and metabolites extracted using ethyl-acetate. Fungal crude extracts were screened for antioxidant activity using 2, 2- diphenyl-1-picrylhydrazyl (DPPH) radical scavenging and reduction of ferric ion assays. Gas chromatography/mass spectrometry (GC/MS) analysis was used to identify the major chemical constituents in active fungal extracts. Results A total of eighteen fungal endophytes with fungal codes CU (061 and 062); ZA (161, 162, 163, and 164); LO (261); CA (041, 042, and 043); FE (081, 082, and 084); GE (091); MO (211 and 212); and NA (021 and 022) were isolated from the eight ethnomedicinal plants A. ornata, A. zygia, A. cordifolia, C. albidum, F. exasperata, G. celosioides, M. thonningii, and N. laevis respectively. ZA 163 and MO 211 fungal extracts showed significant (p < 0.05) radical scavenging activity with IC50 values of 50.53 ± 0.01 and 86.69 ± 0.02 μg/ml respectively. Fungal extract CA 041 demonstrated significantly (p < 0.01) higher iron chelating activity than standard gallic acid with absorbance values of 0.803 and 1.107 at 250 and 500 μg/ml concentrations respectively. Pyrogallol, phenol, 2,6-dimethoxy-, phytol, dl-alpha-tocopherol, alpha-tocospiro, oleamide, methyl stearate, oleic acid, palmitic acid, campesterol, stigmasterol, β-sitosterol, urs-12-en-24-oic acid, 3-oxo-, methyl ester, lup-20(29)-en-3-one, and lupeol were detected in the selected active extracts. Conclusion These results showed that leaves of the selected Nigerian plants harbor diverse group of endophytic fungi, which can be potential antioxidant resource. Graphical abstract


Background
Endophytes are microorganisms that reside within plant tissues and do not cause any deleterious effect to the plants. These microorganisms consist of bacterial and fungal communities that colonize and spend the whole or part of their life cycle inside tissues of the host, without causing noticeable symptoms of plant diseases [1,2]. Endophytic fungi are found in almost plant parts and act as chemical synthesizers inside the host plant, producing a wide range of bioactive secondary metabolites [3,4].
Production of reactive oxygen species known as free radicals occurs frequently in all cells as part of normal metabolic process. However, free radicals have been implicated in the pathogenesis of quite a number of chronic diseases such as diabetes, neurological disorders, and cancer [16]. Free radical scavenging property of antioxidant agents inhibits or delays cellular damage. Dietary intake as well as adequate amount of exogenous antioxidants would prevent the pathological conditions induced by free radicals [17]. However, the use of synthesized antioxidants in the prevention of free radical damage may exude toxic side effects thus the search for natural antioxidant agents is required [18,19]. Endophytic fungi can synthesize secondary metabolites with antioxidant activity that can interrupt the chain reaction of reactive oxygen species [20,21].
Medicinal plants growing in natural habitats are promising hosts of endophytic fungi that might produce bioactive secondary metabolites of pharmaceutical relevance. Different plant parts, especially the leaves, stems, and roots are considered an enormous repository of these fungal endophytes with reported cytotoxic, antifungal, antiviral, and antimicrobial activities [22][23][24]. The genetic resources of these plants found in Nigeria are a veritable source of pharmaceuticals and therapeutics with significant information on their phytochemistry. Additionally, these plants are used

Isolation of endophytic fungi
The collected plant samples were surface sterilized according to the method previously described [25,26] with modifications. To remove dust and debris, samples were washed thoroughly under running tap water, followed by washing in distilled water. The leaf samples were cut into small segments measuring 2-4 mm using sterile razor blades. Surface sterilization of leaf segments was carried out by sequential immersion in 70% ethanol for 60 s, 0.5% sodium hypochlorite for 5 min, 70% ethanol for 30 s, followed by a final rinse in sterilized distilled water for 5 min. Sterilized leaf segments were air-dried aseptically and placed on petri dishes containing potato dextrose agar amended with 0.01% gentamycin antibiotic for the direct contact of cut edges with the nutrient media. Three segments of each plant sample were placed on PDA plates, sealed with parafilm and incubated at 27°C for 5-7 days. Samples were checked daily for visual growth of fungi.

Purification and preservation of endophytic fungi
Following the incubation period, different fungal strains emerged from each sample and individual strains were isolated by transferring hyphal tips onto a fresh PDA medium. This process was repeated several times until a pure endophytic fungal strain with uniform colony was obtained. Pure endophytic fungi isolates were transferred separately to PDA slants and 15% (v/v) sterilized glycerol solution. Growth of endophytic fungi strains in both media was observed for 3-5 days. Glycerol stock solution and PDA slants were maintained at − 20°C and 4°C respectively till further use.

Molecular identification
Identification of endophytic fungi was performed using molecular characterization and sequencing of the internal transcribed spacer region (ITS).

Endophytic fungi DNA extraction
Deoxyribonucleic acid (DNA) extraction of fungal strains was done using Zymo fungal / bacteria DNA extraction kit (Zymo Research Corp., South Africa) according to the manufacturer's instructions. Briefly, pure endophytic fungi strains isolated from the plant samples were grown in potato dextrose broth (PDB) for 6-7 days. Following the formation of endophytes colony, fungal mycelium was drained and homogenized in 200 μL of phosphate buffered saline to aid lysis. About 50 mg of homogenate was harvested into a 1.5 ml microcentrifuge tube and 750 μL lysis solution was added to the tube. The tube was vortex vigorously for 30-60 s and 400 μL of supernatant was transferred into a Zymo-Spin™ IV spin filter in a collection tube and centrifuged at 10,000 rpm for 1 min. After centrifugation, 1200 μL of Fungal/Bacterial DNA binding buffer was added to filtrate in the collection tube. 800 μL of the mixture was then transferred to a Zymo-Spin™ IIC column in a collection tube. The reaction mixture was centrifuged at 10,000 rpm for 1 min. The flow through was discarded and the remaining mixture was centrifuged again at 10,000 rpm for 1 min in the same tube. The column was pre-washed with 200 μL DNA Pre-Wash buffer and centrifuged at 10,000 rpm for 1 min. This was followed by a wash with 500 μL Fungal/Bacterial DNA Wash Buffer and centrifuged at 10,000 rpm for 1 min. The DNA was eluted from the Zymo-Spin™ IIC column to a sterile 1.5 ml microcentrifuge tube using 100 μL of DNA Elution Buffer. The purity and concentration of extracted DNA was evaluated using a NANODROP (ND 1000) spectrophotometer (Thermo Scientific, USA).

Amplification and sequencing
Polymerase chain reaction was carried out to amplify the ITS gene of specific DNA of each fungus using the primer pair ITS-1 (5′-TCCGTAGGTGAACCTGCGG) and ITS-4 (5′-TCCTCCGCTTATTGATATGC) as previously described [27,28]. PCR was done using the Solis Biodyne 5x Hot FIREPol® Master Mix Ready to Load. PCR reaction was performed in a total volume of 20 μL containing 1 x blend master mix buffer, 1.5 mM MgCl 2 , 200 μM of each deoxynucleoside triphosphate (dNTP), 25 pmol ITS1 and ITS4, 2 units of hot FIREPol DNA polymerase and 10-200 ng of DNA. Sterile distilled water was used to make up the reaction mixture. PCR amplification was performed in PTC 100 Peltier Thermal Cycler (MJ Research) using the following protocols: denaturation at 95°C for 15 min; 35 amplification cycles at 95°C for 30 s; primer-specific annealing at 58°C for 1 min and elongation at 72°C for 90 s; a final elongation at 72°C for 10 min. Amplified DNA was checked by electrophoresis at 80 V for 90 min in a 1.5% agarose gel stained with ethidium bromide. The band to PCR product was visualized with a photodocumentation system (Cleaver Scientific, UK).
The amplified products were purified with exo sap and sequenced by Epoch Life science, USA using Sanger sequencing method. Identification of endophytic fungi was performed on the basis of similarity of amplified sequence with ITS sequence data from strains available in the US National Centre for Biotechnology Information (NCBI) database using Basic Local Alignment Search Tool (BLAST) N sequence match routines.

Fermentation and extraction of isolated fungal metabolites
Cultivation of isolated fungal endophytes was carried out in a conical flask containing sterilized rice medium as previously described [25,29] with modifications. Approximately 200 g of rice was soaked in 200 ml sterile water for 10 min in a glass bottle. The flask with its content was autoclaved at 121°C for 20 min and allowed to cool. Three to four pieces of pure mycelia agar plugs were inoculated aseptically into the cooled solid rice medium. A flask of autoclaved solid rice medium without inoculum served as the control. Fungal strain was allowed to grow on rice medium at room temperature (27 ± 2°C) for 4-6 weeks with a routine check. After the incubation period, about 500 ml of ethyl-acetate was added into the culture flask and left overnight at room temperature. The crude ethyl-acetate fungal mixture was filtered through a Whatman filter paper No. 1 under vacuum using a Buchner funnel. Collected supernatant was evaporated to dryness under vacuum on a rotary evaporator (Buchi, Switzerland) at 40°C to obtain crude fungal extract.
In vitro antioxidant activity of fungal extracts DPPH radical scavenging activity Free radical scavenging activities of fungal extracts were measured using 2, 2-diphenyl-1-picryl-hydrazyl (DPPH) as previously described [30,31]. Briefly, 2 ml of varying concentrations (62.5-500 μg/ml) of fungal extracts and positive standard (ascorbic acid) prepared in methanol was mixed with 2 ml of 0.135 mM DPPH in methanol. The mixture was shaken vigorously and incubated in the dark at room temperature for 30 min. Mixture of methanol and DPPH without fungal extract served as the control. Absorbance of samples was measured at 517 nm against the blank tube that contains methanol and used to maintain zero of the UV-vis spectrophotometer. The assay was carried out in three replicates. Inhibition of DPPH was calculated as a percentage of radical scavenging activity of each fungal extract using the formula: Percentage of radical scavenging activity = [(A control -A sample )/A control ] × 100.
Where A is the absorbance reading. IC 50 value (μg/mL) was calculated from percentage of radical scavenging activity against extract concentrations.

Reduction of Fe 3+ ions by ortho-phenanthroline
O-phenanthroline assay was used to determine the reducing capacity of fungal extracts as previously described [32,33]. A reaction mixture containing 1 ml of 2 mM phenanthroline, 2 ml of 0.2 mM ferric chloride hexahydrate (FeCl 3 .6H 2 O), and 2 ml of different concentrations (62.5-500 μg/ml) of fungal extracts was shaken and incubated at ambient temperature for 10 min. Absorbance was measured at 510 nm using a UV-vis spectrophotometer. Gallic acid served as positive standard.

Identification of bioactive constituents by GC/MS
Gas chromatography/mass spectrometry (GC/MS) analysis of bioactive fungal extracts was performed using Agilent 7820A gas chromatograph coupled to an Agilent 5975C inert mass selective detector (MSD) with triple axis detector operated in an electron impact (EI) mode with ionization energy of 70 eV. An HP-5 capillary column coated with 5% phenyl methyl siloxane (30 m × 0.32 mm diameter × 0.25 μm film thickness) was used for the separation. Sample preparation was performed according to the procedure described [34]. The sample (1 μL, diluted 1: 100 in dichloromethane) was injected in splitless mode at an injection temperature of 300°C. Purge flow to split vent was 15 ml/min at 0.75 min with a total flow of 16.654 ml/ min. Helium was used as the carrier gas at the flow rate of 1.487 ml/min with initial nominal pressure of 1.4902 psi and an average velocity of 44.22 cm/sec. The oven temperature was initially programmed at 40°C for 1 min then ramped at 12°C / min to 300°C for 10 min. Run time was 32.667 min with a hold time of 5°C / min. Identification of the chromatographic peaks was based on comparisons of their relative retention times and mass spectra with those obtained from the NIST14.L library data.

Statistical analysis
All assays were carried out in three replicates and results are expressed as mean ± standard error of mean (SEM). IC 50 values were obtained by interpolations from standard curves. Data were analyzed using Graph Pad Prism 8.

Extraction of fungal metabolites
All the fungi isolates were cultivated on solid media and extracted with ethyl-acetate to afford fungal extracts of varying yield, which range from 44 to 2002 mg per 200 g rice medium (Table 2).

Discussion
In this study, eighteen endophytic fungi were successfully isolated from surface sterilized fresh leaf samples of eight medicinal plants. To the best of our knowledge, this is the first report describing the isolation of endophytic fungi residing in the leaf cells of the selected plant samples. After size separation by agarose gel electrophoresis of the ITS PCR products, the PCR products of the plant samples exhibited DNA band size range of 400-700 bp. Detailed taxonomic assignment of fungal isolates remain unresolved due to limitations inherent in fungal ITS sequences. The results obtained from this study thus confirmed that leaves of the medicinal plants: Acalypha ornata, Albizia zygia, Alchornea cordifolia Chrysophyllum albidum, Ficus exasperata, Gomphrena celosioides, Millettia thonningii, and Newbouldia laevis are host to endophytic fungi.
The crude fungal extracts showed vary degrees of antioxidant activity in the DPPH radical scavenging and reduction of ferric ion assays. DPPH (2, 2-diphenyl-1picryl-hydrazyl) is a stable free radical that produces purple color in methanol. Antioxidant activity of the fungal extracts was measured by discoloration to yellow color following the formation of non-radical (2,2-diphenyl-1-hydrazine) molecule [17]. Fungal extracts ZA163 and MO211 exhibited significant (p < 0.05) DPPH radical scavenging activity.
The metal chelating capacity of a compound may also serve as a significant indicator of its potential antioxidant activity [35]. The iron chelating activity of all fungal extracts was determined by reaction with ortho-phenanthroline. Fe 3+ is reduced to Fe 2+ by an antioxidant and the formed Fe 2+ rapidly react with phenanthroline to form a stable red orange colored complex [33,36] [4,[37][38][39][40][41]. Our results suggest that endophytic fungi that reside in the leaves of A. ornata, A. zygia, A. cordifolia, C. albidum, F. exasperata, G. celosioides, M. thonningii, and N. laevis showed promising antioxidant activity.
Fungal extracts ZA163, MO211, LO 261, FE 082, and FE 084 were among the fungal extracts that exhibited effective antioxidant activity with a substantial yield of extract, thus were selected for phytochemical analysis via GC/MS analytical technique. The identified compounds represented phenolic, terpenoid, and sterol classes of secondary metabolites as well as tocopherols and fatty acids. Most of these chemical constituents have been reported to demonstrate remarkable antioxidant activity. Phenolic compounds act as natural antioxidants that exert therapeutic effects such as anti-inflammatory, antidiabetic, antimicrobial, antiviral and vasodilatory effects and prevent various forms of diseases [42][43][44]. Tocopherol plays an important role as a lipid antioxidant in stabilizing subcellular membranes [45]. Fatty acids are able to reduce oxidative stress from free radicals by exerting an antioxidant role [46,47]. Pyrogallol (phenol), alpha tocospiro (tocopherol), and oleamide (amide derivative of fatty acid) are present as the major components in the investigated endophytic fungi extracts. Previous studies have reported the isolation and characterization of graphislactone A, a phenolic benzopyranone antioxidant compound from the endophytic fungus Cephalosporium sp. inhabiting the medicinal plant Trachelospermum jasminoides [11]. Similarly, isolation and characterization of pestacin and isopestacin, a coumarone antioxidant and antifungal agents from the endophytic fungus Pestalotiopsis microspora that resides in the medicinal plant Terminalia morobensis was reported in literature [12,13]. These results suggest that these compounds and others identified in fungal extracts ZA163 from Albizia zygia, MO211 from Millettia thonningii, LO 261 from Alchornea cordifolia, FE 082 and FE 084 from Ficus exasperata could be responsible for the antioxidant activity demonstrated by the endophytes.

Conclusion
The results obtained from this study established that endophytic fungi isolated from medicinal plants: Acalypha ornata, Albizia zygia, Alchornea cordifolia, Chrysophyllum albidum, Ficus exasperata, Gomphrena celosioides, Millettia thonningii, and Newbouldia laevis commonly used in Nigeria local herbal remedies may be a potential source of bioactive compounds, which may be used for the development of antioxidant drugs. Phytochemical analysis revealed the presence of phenols, tocopherols, sterols, terpenoids, and fatty acids that may have contributed to the observed effect. These findings indicate that endophytic fungi hold promise for large scale production of free radical scavenging agents, which can be developed as drug molecules used in the intervention of oxidative stress-mediated diseases.