Introduction

Ocimum is a huge genus within the family Lamiaceae where this family contains more than 150 species that have a long history of traditional uses [1]. Some species of the genus Ocimum are used to treat different types of diseases, especially the species Ocimum basilicum [2,3,4]. Ocimum basilicum is used as an antibacterial, antifungal, and antioxidant [5]. Fresh leaves are used in case of fevers, abdominal, and poor digestion. Leaf’s decoction is a helpful remedy in the treatment of respiratory disorders and promotes the expulsion of kidney stones.

Fungal endophytes live in healthy plant tissues without harmful effects to their hosts [6]. The symbiotic relationship between fungal endophytes and plants has mutual benefits; host plants supply necessary nutrients to the endophytes. Moreover, fungal endophytes assist host plants by preventing invasion of plant pathogens and/or parasites and improving resistance and tolerance of host plants to major biotic or abiotic factors [7]. Endophytes produce bioactive compounds like those in the host plant including alkaloids, coumarins, flavonoids, glycosides, lignans, terpenoids, phenylpropanoids, saponins, quinones, and xanthones [8]. These metabolites can protect their hosts from biological agents and environmental conditions and produce pharmacologically active compounds [9]. On the other side, secondary metabolites of fungal endophytes from Ocimum basilicum are antibacterial, antifungal, antiviral, antioxidants, and anticancer agents [10,11,12,13,14,15]. Recently, bacterial infections associated with antibiotic-resistant strains remain an issue of severe public health concern. To address this problem, there is a need to search and develop new and highly effective antimicrobial agents. To date, extensive data has been generated on the occurrence of bioactive compounds from medicinal plant extracts, while very little exists on the isolation and characterization of secondary metabolites from endophytic fungi [11]. Therefore, the extraction of antimicrobials from new fungal endophytes is required to combat antimicrobial resistance [16, 17]. Therefore, this study aimed to isolate fungal endophytes from Ocimum basilicum leaves and to analyze their secondary metabolites through phytochemical screening and GC–MS. Moreover, to evaluate their antimicrobial activity against human pathogens, cytotoxicity, as well as antioxidant activity.

to assess the preliminary qualitative phytochemical screening of secondary metabo-

lites.

Materials and Methods

Source of Plant sample

The Ocimum basilicum plant used in this study was collected during July 2020. Healthy plants were collected and brought to the laboratory in sterile polyethylene bags in an icebox (4℃).

Isolation of Endophytic Fungi

Isolation of fungal endophytes was performed according to [7]. Leaves of healthy Ocimum basilicum were washed and sterilized with tap water, 70% ethanol, and 4% NaOCl. The sterilized plant parts were cultivated on potato dextrose agar medium (PDA) (Oxoid) supplemented with chloramphenicol (0.2 g/L). The plates were incubated at 27℃ ± 2℃ for 21 days and were observed daily under a stereomicroscope. Hyphal tips that arise out from the cultivated leaf segments were sub-cultured into a PDA medium.

Phenotypic and Genotypic Identification of Endophytic Fungi

Fungal isolates were identified morphologically according to previous studies [18,19,20,21]. Morphological features were observed, which include the color, texture, and diameter of colonies; in addition, vegetative and reproductive structures of the fungi were also recorded. Then endophytes were characterized using molecular identification technique using ITS genes [22].

Extraction of Active Compounds from the Fungal Filtrate

Endophytic fungi were cultured in potato dextrose broth medium (PDB) (Oxoid) at 27℃ ± 2℃ for 21 days under static conditions. The fermentation broth was subjected to filtration under septic conditions to remove fungal mycelia. Culture filtrates of the isolated fungal endophytes were extracted twice using ethyl acetate (EtOAc) (1:1); 100 mL from each filtrate was mixed with 100 mL of ethyl acetate and placed on a vortex shaker for 10 min and settled down for 5 min until the two clear separate layers were formed. The organic layer (EtOAc) was separated from the aqueous layer by the separating funnel. The collected organic phase was evaporated under reduced pressure at 40–45℃ using a rotary evaporator (Heidolph VV2001, Germany); DMSO at 1 mg/mL of concentration was used to dissolve the fungal crude extract and then stored at − 20℃ until further experiments [23].

Qualitative Screening of Phytochemicals

Qualitative screening of the following phytochemicals was performed according to Sarkar et al. (2020) using the following different standard methods [16, 24].

Test for Alkaloids

Crude extracts of endophytic fungi were tested for alkaloids production using Wagner’s reagent. A fraction of the extract was treated with 3–5 drops of Wagner’s reagent [1.27 g of iodine and 2 g of potassium iodide in 100 mL of water] and observed for the formation of a reddish-brown precipitate (or coloration) [25].

Test for Flavonoids

Crude extracts of endophytic fungi were treated with diluted NaOH, followed by the addition of diluted HCl; solubility and color were noted. A yellow solution with NaOH, which turns colorless with diluted HCl, confirms flavonoids Onwukaeme, Ikuegbvweha, and Asonye [26].

Tests for Glycoside, Steroid, and Terpenoids

Qualitative production of glycoside steroids and terpenoids of crude extracts was performed according to methods used by [27] as follows.

For glycosides, 5 mL of each extract was treated with 2 mL of glacial acetic acid in a test tube and a drop of ferric chloride solution was added to it. This was carefully underlaid with 1 mL of concentrated sulfuric acid. A brown ring at the interface indicated the presence of deoxysugar characteristic of cardenolides. A violet ring may appear below the ring, while in the acetic acid layer, a greenish ring may form.

For steroids, 1 mL of chloroform along with few amounts (1 mL) of the sulfuric acid (98%) was mixed with different extracts (1 mL). The existence of steroids was indicated through the appearance of a brown ring in the tube.

For terpenoids, 1 mL of chloroform was added to 2 mL of each extract, followed by a few drops of concentrated sulfuric acid. A reddish-brown precipitate produced immediately indicated the presence of terpenoids.

Test for Tannin

This test was carried out according to the method used by Auwal et al. [28]. Two milliliters (2 mL) of the aqueous solution of the extract was added to a few drops of 10% Ferric chloride solution (light yellow). The occurrence of the blackish-blue color showed the presence of gallic tannins, and a green-blackish color indicated the presence of catechol tannins.

Test for Saponins

Fungal extract (50 mg) dissolved in 5 mL distilled water up to 20 mL water in the tube and the solution shacked for 15–20 min. The formation of a foam layer up to 2 cm or more confirms the presence of saponins [29].

Test for Reducing Sugars

A 2 mL of fungal extract was treated with Fehling solution and heated over a water bath (5–10 min). Brick red precipitate confirms the presence of reducing sugars.

Gas Chromatography-Mass Spectroscopy (GC–MS) Analysis

The metabolites present in the extracts of the endophytic fungal isolates were analyzed, counted, and identified using GC–MS, as explained by Zothanpuia, Passari, Chandra, Leo, Mishra, Kumar, and Singh [30] with minor modifications. Briefly, crude extract of the strain was dissolved in spectroscopy-grade methanol. GC–MS analysis was performed on Trace GC1310-ISQ mass spectrometer (Thermo Scientific, Austin, Texas, USA), using a direct capillary column (length 30 m, thickness 0.25 µm, internal diameter 25 mm). The oven temperature was started at 50℃ held for 5 min and ramped at 5℃ per min up to 230℃ and held for 2 min; 1 μL of the sample was injected at 250℃ using helium as a carrier gas, split at the ratio of 1:30. The mass spectrometer was run in the electron ionization (EI) mode at 200℃ and 70 eV with a scan range of 40 to 1000 m/z. The spectrum of the detected compounds was compared with the spectrum of the known compounds stored in the WILEY 09 (Wiley, New York, NY, USA) and NIST 11 library. The name, molecular weight, and chemical structure of the detected compounds were also determined.

Antimicrobial Activity

The antimicrobial activity of ethyl acetate crude extracts of fungal isolates was evaluated on Muller Hinton agar (MHA, India) for bacteria and PDA for yeast. Twenty-four hours young culture of Staphylococcus aureus ATCC 6538, Bacillus cereus ATCC 10,987, Bacillus subtilis ATCC 6633, Escherichia coli ATCC 8739, Salmonella typhimurium ATCC14028, Klebsiella pneumonia ATCC 13,883, and Pseudomonas aeruginosa ATCC 9072 and 48 h young culture of Candida albicans ATCC10231 were cultured on the surface of prepared MHA and PDA for bacteria and fungi, respectively. Wells (6 mm) were cut using a sterile cork borer; 100 µl of crude extracts was transferred to each well individually and left for 2 h at 4℃. Chloramphenicol was used as a control for bacterial strain, while fluconazole was used as a control for Candida albicans, and then, the plates were incubated for 24 h at 37℃ for bacteria and 48 h at 28℃ for Candida albicans. After incubation, the inhibition zones were measured and recorded [31,32,33].

Antioxidant activity

DPPH assay

Antioxidant activity of crude extracts of endophytic fungal isolates was carried out using DPPH (2, 2-diphenyl-1-picrylhydrazyl) method according to Khalil, Abdelaziz, Khaleil, and Hashem [34] with minor modifications. Different concentrations of crude extracts (1000 µg/mL, 500 µg/mL, 250 µg/mL, 125 µg/mL, 62.5 µg/mL, 31.25 µg/mL, 15.62 µg/mL, and 7.81 µg/mL) were used to determine the scavenging of DPPH radicals. Antioxidant activity of standard and extracts was determined as DPPH scavenging activity (%): [((control absorbance − extract absorbance)/(control absorbance)) × 100].

ABTS Assay

Another assay for evaluation antioxidant activity is ABTS ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)). The antioxidant activity of all crude extracts was evaluated using ABTs assay according to the method used by Nicoletta [35] with minor modifications.

In-vitro Cytotoxicity

The cytotoxicity of crude extracts of the endophytic fungal strains at concentrations 500 µg/mL and 1000 µg/mL was determined using the MTT protocol [36] with minor modification against normal Vero and Wi 38 cell lines which collected from ATCC. The viability and inhibition percentages were calculated, as shown in Eqs. 1 and 2, as follows:

$$\mathrm{Viability \%}=\frac{\mathrm{Test OD}}{\mathrm{Control OD}}\times 100$$
(1)
$$\mathrm{Inhibition \%}=100-\mathrm{Viability \%}$$
(2)

Statistical Analysis

The data were expressed as the mean ± SDEV value, which was calculated by using Minitab 18 software extended with a statistical package and Microsoft Excel 365.

Results and Discussion

Isolation and Identification of Endophytic Fungi

Three different fungal isolates were obtained from surface-sterilized Ocimum basilicum plant parts and coded as S-1, S-2, and S-4. Morphological identification illustrated that fungal isolates S-1 were identified as Aspergillus nidulans where colony diameter after 7 days at 28℃ on PDA about 35 mm, blue-gray with the heavy conidial formation and few Cleistothecia, hyaline reverse deeper, and with abundant Cleistothecia surrounded by dull yellow or buff Hull cells. In addition, fungal isolate S-2 was identified morphologically as A. fumigatus, where colonies grow rapidly, reaching 30–50 mm diameter in 4 days at 28℃, on (PDA), smoky gray-green, reverse faint yellow. Furthermore, fungal isolate S-4 was identified as Aspergillus flavus where colonies grow rapidly, reaching 30–50 mm diameter in 4 days at 28℃, on PDA, dark yellowish-green colonies (powdery) – reverse creamy, pale brown, as shown in Fig. 1.

Fig. 1
figure 1

Morphological identification of endophytic fungal strains A. nidulans (A), A. fumigatus (B), and A. flavus (C): (1) surface of culture on PDA; (2) reverse color of culture; (3) conidiophore and head under the light microscope (400X); (4) conidia under the light microscope (800X)

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Phylogenetic trees revealed that three fungal strains were identified genetically as A. nidulans, A. fumigatus, and A. flavus which are related to fungal strains A. nidulans MG518452.1, A. fumigatus MT529212.1, and A. flavus KY964055.1 that were deposited in the NCBI database with similarity percentages of 97.08%, 90.86%, and 94.41%, respectively (Fig. 2). Three fungal strains A. nidulans, A. fumigatus, and A. flavus were recorded in gene bank with accession numbers MZ045561, MZ045562, and MZ045563, respectively.

Fig. 2
figure 2

Phylogenetic tree of genetically identified A. nidulans, A. fumigatus, and A. flavus with accession numbers MZ045561, MZ045562, and MZ045563, respectively

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Phytochemical Analysis

The presence of secondary metabolites indicates the importance of natural products as therapeutic agents. Fungal endophytes are a rich source for many other secondary metabolites such as antibacterial, antifungal, anticancer, and antiparasitic compounds. Different endophytic aspergilli have the ability to produce promising secondary metabolites including alkaloids, terpenoids, ρ-terphenyls, diphenyl ether cytochalasins, xanthones, phenalenones, sterols, and anthraquinone derivatives with different biological activities [37]. The endophytic fungi A. nidulans, A. fumigatus, and A. flavus exhibited antimicrobial activity due to the production of several secondary metabolites. The fungal endophytes from leaves of Ocimum basilicum have promising antimicrobial activity against Gram-positive, Gram-negative bacteria and C. albicans. The results of the preliminary phytochemical study were given in Table 1 and indicated some secondary metabolites found in ethyl acetate fungal extract. Glycosides play an important role against predation by microorganisms, insects, and herbivores [38]. Steroids are known to have antibacterial properties; steroidal compounds specifically interact with membrane lipid and causing leakages of liposomes [39]. Moreover, terpenoids are a cell wall inhibitors class of phytochemicals with antimicrobial properties which cause membrane disruption [40]. Eventually, saponins can affect bacterial growth is due to their ability to cause leakage of proteins and certain enzymes from the cell [41].

Table 1 Phytochemical screening of endophytic A. nidulans, A. fumigatus, and A. flavus metabolites
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GC–MS

GC–MS analysis gives a representative spectral output of each one of the compounds found in the analyzed samples. So, in the past few years, GC–MS has become well recognized as a major technology platform for describing the secondary metabolites in both plant and non-plant species [42, 43]. The GC–MS chromatograms (Fig. 3) of ethyl acetate crude extracts of the fungal strains and the data listed in Table 2 illustrated the presence of 22, 22, and 20 compounds in the extracts of A. nidulans, A. fumigatus, and A. flavus, respectively. The results also revealed the existence of 16 compounds with the same name, molecular weight, and chemical structure at the same retention time (RT) with different peak area % in the extracts of the three fungal strains. The major compounds in the fungal extracts are bisabolol oxide B; hexadecanoic acid methyl ester; 9,12-octadecadienoic acid (Z, Z)- methyl ester; 9-octadecenoic acid (Z)- methyl ester; linoelaidic acid; trans-13-octadecenoic acid; hexadecanoic acid 2-hydroxy-1-(hydroxymethyl)ethyl ester; glycidyl palmitate; 9,12-octadecadienoic acid (Z, Z)- 2-hydroxy-1-(hydroxymethyl)ethyl ester; 9,17-octadecadienal, (Z); ethyl (9z,12z)-9,12-octadecadienoate; glycidyl oleate; and linoleoyl chloride. These compounds have different biological activities such as antimutagenic, anti-inflammatory, antiseptic, anticancer, antihistaminic, anti-corona, antiarthritic, anti-eczemic, antioxidant, antimicrobial, antidiarrheal, antiproliferative, nematocidal, pesticide, hypocholesterolemic, hepatoprotective, hemolytic, and immune function modulation.

Fig. 3
figure 3

GC–MS chromatogram of ethyl acetate crude extracts of A. nidulans, A. fumigatus, and A. flavus

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Table 2 The detected compounds through GC–MS of crude extracts of the fungal strains
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Most of the detected compounds are belonging to fatty acid, fatty acid esters, tetrahydrofurans, and sterols. Different studies were reported that these compounds were extracted from the basil plant (Ocimum basilicum) [68,69,70,71]. Endophytic fungi living in medicinal plants can make the same pharmacological bioactive secondary metabolites in the same way as their host medicinal plants, which have been used for a long time in traditional medicine and even now are utilized for their health advantages [72,73,74]. In addition, some reports mentioned the presence of these metabolites in the extracts of aspergilli and other endophytic fungi isolated from different plants [58, 75,76,77]. A. fumigatus has been produced twenty-nine bioactive metabolites that have antimicrobial activities against human pathogenic bacteria as E. coli, S. aureus, and fungi as Candida albicans [37]. Furthermore, 5-hydroxy methyl furan-3-carboxylic acid and 5-acetoxy methyl furan-3-carboxylic acid as furan derivatives were isolated from the culture of endophytic A. flavus, that exhibited antibacterial activity against Staphylococcus aureus [78].

Antimicrobial Activity

Bioactive compounds produced from medicinal plants and endophytic microbes have high efficacy against pathogens with low toxicity on cells [79]. Data reported in the Table 3 explained that the endophytic fungal strains exhibited promising antimicrobial activity against all tested microbial strains (S. aureus, B. cereus, B. subtilis, E. coli, S. typhimurium, K. pneumonia and P. aeruginosa, and C. albicans ATCC10231). Generally, the zones of inhibition were ranged from 13 to 26 mm in diameter. The crude extract of A. nidulans and A. flavus exhibited the highest antimicrobial activity against the tested bacteria compared to A. fumigatus which exhibited the lowest inhibition zones among them. With the same behavior, the crude extracts of endophytic A. nidulans and A. flavus exhibited considerable antifungal activity against C. albicans with inhibition zones of 22 mm and 23 mm, respectively, compared to the inhibition zones caused by A. fumigatus (19 mm), as shown in Fig. 4.

Table 3 Antimicrobial activity of crude extract of the fungal strains
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Fig. 4
figure 4

Antibacterial activity of ethyl acetate extract of (A = A. nidulans, B = A. fumigatus, C = A. flavus, D = Chloramphenicol) against (K pneumonia (a), P aeruginosa (b), S. typhimurium (c), B. cereus (d), B. subtilis (e), E. coli (f), S. aureus (g), and C. albicans (h))

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This antimicrobial activity may be attributed to several factors as (1) the presence of some major compounds that can inhibit the microbial growth in the crude extract of A. nidulans, A. fumigatus, and A. flavus, according to El-Fayoumy et al. [74]. These compounds included 9-octadecenoic acid (Z)-; methyl ester methyl stearate; 9,12-octadecadienoic acid (Z, Z)-; 2-hydroxy-1-(hydroxymethyl) ethyl ester; and 9,17-octadecadienal, (Z)-, according to GC–MS analysis. (2) The appearance of terpenoids in phytochemical analysis of fungal crude extracts in all crude extracts may also be responsible for antimicrobial activity because it can disrupt the cell membrane of the microbes, as reported by Jasmine et al. [40]. (3) The detection of glycosides in phytochemical analysis of fungal crude extracts that cause cell lysis and disruption of the cytoplasmic membrane leading to loss of membrane selective permeability. This may also share in the antimicrobial activity of the tested crude extracts [80].

Antioxidant Activity

Antioxidants are compounds that defeat reactive oxygen species (ROS) which resulted from biological reactions as a by-product [81]. In addition, antioxidants are capable of holding and balancing the free radicals which cause several diseases [82]. Antioxidants have been considered as therapeutic agents where they possess anti-atherosclerotic, anti-inflammatory, antitumor, anticarcinogenic, antimutagenic, and antimicrobial properties. In our study, the antioxidant activity of crude extracts of endophytic fungal strains A.nidulans, A. fumigatus, and A. flavus at different concentrations (1000–7.81 µg/mL) was performed using DPPH and ABTS methods, as shown in Fig. 5. Results proved that the antioxidant activity of A. fumigatus was the highest among other endophytic fungal strains, followed by A. nidulans and A. flavus. Additionally, IC50 of A. fumigatus was 68.4 µg/mL compared to AA (5.75 µg/mL) in the case of DPPH, while using ABTs IC50 of A. fumigatus and AA were 77.9 µg/mL and 6.6 µg/mL, respectively. On the other hand, IC50 of A. nidulans and A. flavus were 166.3 µg/mL and 347.1 µg/mL and 151.2 µg/mL and 246.3 µg/mL using DPPH and ABTs assays, respectively. Previous studies confirmed the potential antioxidant activity of fungal endophytes [83]. Furthermore, Khalil, Abdelaziz, Khaleil, and Hashem [34] found that A. Ochraceus which was isolated from Avicennia marina has strong antioxidant activity using DPPH free radical method.

Fig. 5
figure 5

Antioxidant activity of crude extracts of endophytic A. nidulans, A. fumigatus, and A. flavus at different concentrations using DPPH (A) and ABTs (B) methods

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Cytotoxicity

In this study, cytotoxicity of ethyl acetate crude extracts of endophytic A. nidulans, A. fumigatus, and A. flavus at concentrations 500 µg/mL and 1000 µg/mL was performed, as shown in Fig. 6. Results showed that endophytic fungal extracts of A. nidulans, A. fumigatus, and A. flavus are safe in use, where cell viability of and Wi 38 normal cell lines was more than 96% after their treatment with the extracts at concentrations from 500 to 1000 µg/mL. Likewise, IC50 of all fungal extracts against Vero and Wi 38 cell lines was greater than 1000 µg/mL; this indicates that these extracts are non-toxic because Ioset, Brun, Wenzler, Kaiser, and Yardley [84] reported that the compound is safe when IC50 is greater than 90 μg/mL. Khalil, Abdelaziz, Khaleil, and Hashem [34] reported that all endophytic fungal strains which isolated from Avicennia marina did not display significant toxicity to Vero normal cell line.

Fig. 6
figure 6

Cytotoxicity of endophytic fungal extracts at concentrations (1000–500 µg/mL) against Vero and Wi 38 normal cell lines

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Conclusion

In the current study, three endophytic fungi were isolated and identified morphologically and genetically as A. nidulans, A. fumigatus, and A. flavus. These endophytic fungi have been shown a powerful source of natural compounds with biological activities. Crude extracts of endophytic A. nidulans, A. fumigatus, and A. flavus have shown promising antibacterial activity against Gram-negative and Gram-positive bacteria as well as antifungal activity against unicellular fungi. Likewise, these endophytic fungal compounds revealed antioxidant activity with no cytotoxic effect on normal cell lines. Therefore, these compounds produced by these endophytic fungi may be considered safe and can become alternatives to commercial antimicrobial agents.