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BY 4.0 license Open Access Published by De Gruyter Open Access November 21, 2023

Evaluation and chemical characterization of bioactive secondary metabolites from endophytic fungi associated with the ethnomedicinal plant Bergenia ciliata

  • Jiwan Raj Prasai , Senthuran Sureshkumar , Waseem Ahmad , Mohammad Ashraf , Chinnadurai Gopi , Pandy Rajapriya , Abeer S. Aloufi , Nandakumar Natarajan and Mohan Pandi EMAIL logo
From the journal Open Chemistry

Abstract

The objective of this work was to assess the bioactive potential of endophytic fungi, Colletotrichum brevisporum (JPSK3), Pestalotiopsis microspora (JPSK19), and Guignardia mangiferae (JPSK25), from Bergenia ciliata. The antibacterial effects were determined by the well diffusion technique against human pathogens (Staphylococcus aureus, Escherichia coli, Bacillus cereus, and Salmonella typhi) and they showed good activities. In the antioxidant assay, the fungal extract P. microspora shows higher free radical scavenging effects in 2,2-diphenyl-1-picrylhydrazyl (82.48%), hydrogen peroxide (84.91%), superoxide (78.85%), and reducing power absorbance of 0.125 at a dosage of 125 g/mL than other two fungi. The fungal extract was also subjected to phenol and flavonoid quantifications. The fungus P. microspora shows the highest phenol (89.56 0.03 mg of GAE/g of extract) and flavonoid (51.52 0.69 mg of quercetin equivalents/g of extract), respectively. The chemical composition of abundant biologically active compounds was determined by gas chromatography-mass spectrophotometry (GC-MS). Furthermore, it was confirmed through spectral and analytical analysis (thin layer chromatography, ultraviolet, Fourier transform infrared spectroscopy, high-performance liquid chromatography, GC-MS, and nuclear magnetic spectroscopy). In P. microspora, the compound phenol, p-tert-butyl (C10H14O), was confirmed for the first time. This fungal compound could be a potential alternative medicine in the future. This is the first work on endophytic fungal studies of ethnomedicinal plant Bergenia ciliata.

Graphical abstract

1 Introduction

Endophytic fungi play a critical role in the promotion of plant growth, stress tolerance, drought resistance, insect and herbivore resistance, and other physiological and ecological roles [1]. Endophytes are microorganisms that colonize within plant tissues but cause no damage or disease symptoms. The current investigation aims to use unexploited locations, herbal plants, and their endophytic fungi to discover new, inexpensive, and efficacious bioactive compounds. This is an attempt to address the significant challenges posed by resistance [2]. Endophytic fungi residue within most living plant host tissues is reported to produce exceptional and novel bioactive compounds [3]. The resource demonstrated its significance and diversity as it provided a wide range of active natural compounds with abundant pharmacological activity [4]. Many secondary metabolites have been extracted and characterized from endophytic fungi, with antimicrobial, antioxidant, anticancer, antitubercular, antiparasitic, and antiviral properties. Hence, these bioactive compounds have been widely applied in pharmaceutical and agricultural applications [5,6].

Endophytic fungal compounds can also be utilized as antimicrobial candidates since they produce different bioactive compounds like alkaloids, flavonoids, terpenoids, phenols, etc. The improvement of novel antibiotics is essential to counteract the higher risk of drug-resistant pathogens [7,8]. Free radicals are reactive oxygen species (ROS) that are accumulated in our bodies due to a variety of biological events that cause different types of harm caused by stress to the membrane-bound biomolecules, which in turn causes numerous diseases [9]. In addition to nutritional consumption, an enormous level of exogenous antioxidants would inhibit the pathological symptoms caused by free radicals [10]. Plants–fungal diversity raises the chances of finding a new compound in the fungal communities [11]. Endophytic fungi with antimicrobial properties are predicted to be isolated due to the ability of these plants to generate bioactive compounds [12].

Despite the existing information on the flora, fauna, and traditional utilization of medicinal plants, notably among the mountain communities of Sikkim, India, the country still represents an underexplored region in terms of its potential as a host for medicinal plants and the abundance of valuable microorganisms. Bergenia ciliata is one of the most precious indigenous plants, which the people commonly used in health care. However, there are no reports of endophytic fungal secondary metabolites from this plant. The current study aimed to investigate the biological potentials, i.e., antioxidant, antibacterial, and phytochemical evaluation of Colletotrichum brevisporum (JPSK3), Pestalotiopsis microspora (JPSK19), and Guignardia mangiferae (JPSK25) isolated from Bergenia ciliate. The chemical profile of the most abundant biologically active metabolites was determined by gas chromatography-mass spectrophotometry (GC-MS) with the NIST library. Furthermore, the bioactive biological compound was confirmed using spectral and analytical techniques like thin layer chromatography (TLC), ultraviolet (UV), Fourier transform-infra red (FT-IR), high-performance liquid chromatography (HPLC), GC-MS, and nuclear magnetic resonance (NMR) spectroscopy.

2 Materials and methods

2.1 Isolation and identification of fungi

Fresh, healthy Bergenia ciliata leaves from the mountainous region of Sikkim, India, were collected on sterile polythene bags, transported to the lab, and processed within 24 h for endophytic fungal isolation. Isolated fungi were identified using morphological and molecular techniques, as previously published in our diversity documentation studies [13].

2.2 Extraction of secondary metabolic compounds

The pure fungal isolates were grown on 500 mL potato dextrose (Hi-media) broth in a 1,000 mL Erlenmeyer flask and were incubated as the static culture at 24 ± 1°C for 3 weeks. After that, the fungi were filtered and the mycelia were separated. Then, an equal amount of ethyl acetate was added to the filtrate, shaken well for 20 min, and kept for 10 min until the two transparent separate immiscible layers developed. The upper layer was collected and dehydrated using a rotary vacuum evaporator (Model no. IKA-RV10). The dry crude compound was collected in small glass vials and was evaluated for biological activities [14].

2.3 Phytochemical analysis

2.3.1 Estimation of total phenolic content (TPC)

The Folin–Ciocalteau reagent-based test was employed to estimate the total phenol levels of ethyl acetate extracts from three distinct endophytic fungi, with gallic acid serving as the standard. Each extract (1 mg/mL) of 100 µL was mixed with 500 µL of 1 N Folin–Ciocalteau reagent. Then, 1.5 mL of 20% NaCO3 was added to the mixture, and the final amount was made to 5 mL by diluting with deionized water. The suspension was incubated for 30 min at 37°C, and absorbance was measured at 760 nm using a UV-vis spectrophotometer. An identical protocol was employed using a 1 mL aliquot of gallic acid at concentrations of 5, 10, 15, and 50 µL as standards for constructing the calibration curve [14].

2.3.2 Estimation of total flavonoid content (TFC)

The quantification of TFC in ethyl acetate extracts derived from three distinct endophytic fungi was conducted using the aluminium chloride colorimetric technique [35]. A 100 µL extract (1 mg/mL) was combined with 2 mL of distilled water and 0.15 mL of a 5% NaNO2 solution. Subsequently, after 6 min, 0.15 mL of a 10% AlCl3 reagent was introduced into the mixture, which was then allowed to stand undisturbed for an additional 6 min. Subsequently, 2 mL of 4% NaOH was introduced. The ultimate volume was increased to 5 mL with the addition of deionized water and allowed to stand for 15 min at ambient temperature. The absorbance was measured at a wavelength of 510 nm by a UV-visible spectrophotometer. Quercetin, a flavonoid, served as the reference compound. The findings were illustrated in terms of quercetin equivalents (QE).

2.4 Antioxidant activity

2.4.1 2,2-Diphenyl-1-picrylhydrazyl (DPPH) assay

For the DPPH assay, aliquots of the fungal extract at various concentrations were mixed with 2 mL of DPPH (0.1 mM). The suspension was mixed well before being incubated for 10 min at 37°C. The resultant suspension absorbance was measured at 517 nm. The radical scavenging capacity was determined using the following formula:

(1) Scavenging effect ( % ) = [ ( A 517 of control A 517 of sample ) / A 517 of control ] × 100 .

2.4.2 Reducing power assay

The reducing power assay was conducted on respective extracts (1 mg/mL) at various dosages. Initially, the extracts were added into 2.5 mL of a 0.2 M phosphate buffer (pH 6.6), followed by the addition of 2.5 mL of a 1% potassium ferricyanide. Following a 20 min incubation at 50°C, 2.5 mL of a 10% solution of trichloroacetic acid was introduced into the suspension. Subsequently, centrifugation was performed at a speed of 3,000 rpm for 10 min. Next, 5 mL of the supernatant was combined with 2.5 mL of distilled water and 0.5 mL of a 0.1% ferric chloride. The measurement of absorbance was conducted at 700 nm using a blank solution as the reference. Vitamin C was employed as a positive control [15].

2.4.3 Superoxide radical scavenging assay

The ability of the fungal extracts to scavenge superoxide anions was assessed by measuring the reduction of nitroblue tetrazolium (NBT) using spectrophotometry [16]. The superoxide anion was produced using a non-enzymatic mechanism. A fungal extract of 0.1 mL was combined with an equivalent volume of 60 µM phenazine methosulphate and 468 µM NBT in a sodium phosphate buffer (pH 7.4). The suspension was incubated for 5 min, after which the absorbance was measured at a wavelength of 560 nm relative to a blank sample. The vitamin-C was employed as a positive control. The percentage of scavenging activities (%) was determined by the following equation:

(2) Scavenging activities ( % ) = [ 1 ( Abs 560 nm of the sample / Abs 560 nm of control ) ] × 100 .

2.4.4 Hydrogen peroxide (H2O2) scavenging assay

The scavenging ability of the extract towards H2O2 radicals was assessed. A saline solution (pH 7.4) was used to prepare a hydrogen peroxide with a concentration of 40 mM. The concentration was evaluated using a UV spectrophotometer by measuring the absorbance at 230 nm. The fungal extract (0.1 mg/mL) was mixed with various concentrations and subsequently added to a solution of hydrogen peroxide. The resulting mixture was then subjected to UV spectrophotometry at a wavelength of 230 nm. A blank solution, consisting of PBS without H2O2, was utilized as a reference to quantify the absorbance. The H2O2 scavenging percentage was calculated for both the crude extract and the standard compound (ascorbic acid) using the following formula [9]:

(3) Scavenging ability ( % ) = [ ( A 230 of control A 230 of sample ) / A 230 of control ] × 100 .

2.5 Analysis of the antibacterial effect of the fungal extract

Antibacterial effects of the fungal extract of three different selected endophytic fungi were tested against four human pathogens (two Gram-positive and two Gram-negative) such as Bacillus cereus (MTCC430), Staphylococcus aureus, (ATCC6518), Escherichia coli (ATCC3739), and Salmonella typhi (MTCC733) by a well diffusion technique. The bacterial pathogen was loaded on nutrient agar medium plates. The wells were made on an agar plate by using a cork borer, and the fungal extracts at concentrations of 25, 50, 75, 100, and 125 µL were poured into separate wells. The positive control was streptomycin (concentration, 50 µL), and dimethyl sulphoxide was utilized as a negative control. All the plates were incubated at 37°C for 24–48 h. After the incubation time, the inhibitory zones were measured. Three replicas were performed for each antibacterial activity test.

2.6 GC-MS analysis

The fungal extracted was studied by GC-MS (Agilent GC7890 MS 5975C) with a Multifunctional Autosampler (AOC-6000), fitted with an RTX-5 (60 m × 0.25 nm × 0.25 µm). The machine was set to an initial temperature of 50°C for 2 min. During this time, the oven temperature was augmented up to 280°C, increasing at a rate of 5°C/min for 9 min. The ionization voltage was 70 eV, and helium gas was used as a carrier at a flow rate of 1 mL/min with an injection amount of 2 µL (spit ratio, 10:1). The injection port base temperature was regulated at 260°C. The mass spectral scan range was fixed at 45–450 (m/z). The unknown compounds in the fungal extract were determined by searching the mass spectra with the known database of NIST05 (National Institute of Standards and Technology, USA) libraries. Further, the name, molecular weight, and structure of the compound of the test materials were determined [17].

2.7 TLC analysis

The fungal extract was suspended in methanol and utilized for TLC analysis on silica-coated plates (Silica gel GF254, Merck, Germany). The solvent chloroform/methanol (9:0.2) was used as a mobile phase. After that, plates were dehydrated and observed under UV light at 254 and 366 nm [18]. The R f value was determined by the following equation:

R f = distance travelled by the compound (solute)/distance travelled by the solvent front.

2.8 UV spectrophotometer and FT-IR spectroscopy analysis.

After chromatography, the travelled area of the TLC plate with the putative fungal compound was discarded with the utmost care by scraping off the silica at a suitable R f value and thoroughly eluting and dissolving it in methanol. The separated fungal compound was suspended in 100% methanol, and determined using a Beackman-40 spectrophotometer and compared with standard compounds.

The extracted compounds were analysed using FT-IR spectroscopy on a Shimadzu FT-IR 8000 series machine. The separated fungal compounds were ground with IR grade KBr (1:10), pressed into discs under vacuum using a spectra lab pelletizer, and compared with standard compounds. A single functional group and various lateral chains and hydrogen bonds were recognized. The IR spectrum of the functional group was recorded in the wave number range between 4,000 and 500 cm−1.

2.9 HPLC

The fungal compound was subjected to HPLC analysis. A Dionex P580 HPLC system fitted with a photodiode array detector (UVD340S, Dionex Softron GmbH, Germering, Germany) was employed in this study. The isolation column (125 × 4 mm length × internal diameter) was prefilled with Eurospher-10 C18 (Knauer, Germany), and a gradient of nanopure water (pH 2) and methanol was employed as an eluent. A fungal extract (2 mg) was reconstituted with 2 mL of methanol of HPLC grade. The resulting mixture underwent sonication for 10 min, followed by centrifugation at 3,000 rpm for 5 min. Then, 100 μL of the sample was transferred to the vial with 500 μL of methanol, and with the help of a micro-syringe, the sample volume was injected into the HPLC machine. Prior to applying into the column, the samples and mobile phase were filtered using a 0.2 µm nylon membrane filter. The absorption peaks and retention time (RT) of the compounds were noted on a UV spectrophotometer at 280 nm.

2.10 NMR spectroscopy analyses of ¹H and ¹³C

The 1H NMR spectrum was recorded using a BRUKER NMR ADVANCE spectrometer operating at 400 MHz for 1H and 400 MHz for 13C. The samples were suspended in deuterated chloroform (CDCl3) as a solvent and were identified by comparing chemical shifts and coupling constants with related compounds. Chemical displacements were reported as δ-relative values (δ = 0.0 ppm) to tetramethylsilane as an internal reference and the coupling constants were reported in hertz. The accelerating voltage was 10 kV and the spectra were obtained at 37°C.

2.11 Statistical analysis

All the tests were conducted in triplicate, and values are given as mean ± SD. The statistical studies were conducted using one-way ANOVA. The P values < 0.05 were considered significant, analysed by Tukey’s post hoc assay. The Microsoft Excel version 2010 was utilized for determining mean ± SD.

3 Results and discussion

In this study, Bergenia ciliata associated endophytic fungi, C. brevisporum (JPSK3), P. microspora (JPSK19), and G. mangiferae (JPSK25) (Figure 1) were used to screen secondary metabolites. The metabolites were evaluated for their bioactive potentials such as phytochemicals, and antibacterial and antioxidant properties. Further, the bioactive compounds were characterized using different spectral and analytical methods.

Figure 1 
               Cultural morphology and microscopic spores (40×) observation of selected fungal endophytes Colletotrichum brevisporium (JPSK3), Pestalotiopsis microspora (JPSK19), and Guignardia mangiferae (JPSK25).
Figure 1

Cultural morphology and microscopic spores (40×) observation of selected fungal endophytes Colletotrichum brevisporium (JPSK3), Pestalotiopsis microspora (JPSK19), and Guignardia mangiferae (JPSK25).

3.1 Analysis of total phenol and flavonoid contents

Phenols and flavonoids are well-known antioxidants and other biologically important agents. Many studies are now focusing on phenol and flavonoid metabolites from fungal species due to their multiple health benefits in humans [19,20,21]. Numerous works have highlighted phenol and flavonoid secondary metabolites in endophytic fungi [9,22]. In the present study, phenol and flavonoid compounds reported from three endophytic fungi, C. brevisporum (JPSK3), P. microspora (JPSK19), and G. mangiferae (JPSK25), are considered. All three fungi produced phenol and flavonoid metabolites ranging from 60.59 ± 0.04 to 89.56 ± 0.03 mg of GAE/g and 31.55 ± 0.70 to 51.52 ± 0.69 mg of QE/g, respectively. Among the three, P. microspora (JPSK19) produced the highest amount of phenol content (89.56 ± 0.03 mg of GAE/g of the extract) and flavonoid (51.52 ± 0.69 mg of QE/g of the extract), when compared to other two fungi G. mangiferae (80.44 ± 0.19 mg GAE/g, 37.53 ± 0.67 mg of QE/g) and C. brevisporum (60.59 ± 0.04 mg QE/g, 31.55 ± 0.70 mg of QE/g), as shown in Table 1. In line with this work, the ethyl extract of the Aspergillus terreus from the leaves of Hibiscus sabdariffa shows a phenol content of 204.5 ± 0.4 mg GAE/g [23]. According to Rai et al. [24], the ethyl acetate extract of eight different endophytic fungi from the medicinal plant of Oroxylum indicum (L.) produced phenol and flavonoid contents in various concentrations. Among the eight fungi, C. gloeosporioides shows higher amounts of phenol and flavonoid contents (55.16 and 81.95 mg GAE/g, respectively). Six fungi that produce flavonoids were found in the Conyza blinii H. medicinal plant, according to Tang et al. [25]. When compared to other strains, the CBL11 strain gives a higher yield of flavonoids. In this present study, phenol and flavonoids were screened from three different fungal strains isolated from Bergenia ciliata.

Table 1

Phenol and flavonoid contents

Sl. no. Fungal extract TPC (mg of GAE/g of extract) TFC (mg of QE/g of extract)
1 Colletotrichum brevisporum (JPSK3) 60.59 ± 0.04* 31.55 ± 0.70#
2 Pestalotiopsis microspora (JPSK19) 89.56 ± 0.03* 51.52 ± 0.69#
3 Guignardia mangiferae (JPSK25) 80.44 ± 0.19* 37.53 ± 0.67#

Note: Data were depicted as mean ± SD (n = 3). Mean data followed by different superscripts (“*” and “#”) denote significant statistical differences (P < 0.05).

3.2 Antioxidant assay

Radical scavenging actions are crucial because they neutralize free radicals’ harmful effects on food and biological systems. Currently, various techniques are utilized to determine the antioxidant compounds present in fungal sources. Chemical studies are based on the capacity to neutralize free radicals. The oxidation end-point was identified [26], using numerous radical-producing devices and methodologies. In this work, ethyl acetate extracts of three distinct fungal compounds were tested for antioxidant effects such as DPPH, H2O2, O2˙, and reducing power assay. Each fungal extract has potent antioxidant properties.

3.2.1 DPPH assay

The DPPH scavenging model is an important tool for assessing antioxidants’ ability to remove free radicals. In the presence of an odd electron, the DPPH radical turns deep purple. When an antioxidant molecule provides an electron to DPPH, it decolorizes, which is determined by comparing the absorbance at 517 nm [27,28]. In the present work, the antioxidant activity of ethyl acetate extracts (concentration, 25–125 µg/mL) of the three fungi C. brevisporum (JPSK3), P. microspora (JPSK19), and G. mangiferae (JPSK25) was determined. The fungal extracts show significant radical scavenging activity at all concentrations. Among the three fungi, P. microspora exhibited a high scavenging activity of 82.48%, while C. brevisporum and G. mangiferae showed moderate antioxidant activity of 56.84 and 66.67%, respectively, at a concentration of 125 µg/mL. Vitamin C was utilized as a standard drug and their free radical scavenging activity was found to be 89.74%, as shown in Figure 2a. All three fungal extracts exhibit moderate to high antioxidant activity with lower IC50 values, indicating the stronger antioxidant activity of fungal compounds. P. microspora exhibits significant DPPH activities at an IC50 concentration of 61.21 ± 2.92 µg/mL when compared to the other two fungi, as shown in Table 2. According to Hidayat et al. [29], two endophytic fungi Apodus oryzae (R2MC3A) and Diaporthe sp. (P1DS1C) from agarwood tree show significant antioxidant activity (IC50 of 60.92 and 76.65 mg/mL), respectively, which very well correlates with our reports. Likewise, Gunasekaran et al. [30] highlighted that the endophytic fungus Alternaria sp. (ML4) separated from Mussaenda luteola L. shows a high percentage of antioxidant activity (85.20%) at a concentration of 300 µg/mL.

Figure 2 
                     Antioxidant activities of the fungal extract (a) DPPH and (b) H2O2. (c) Power reducing and (d) superoxide radical assay. C. brevisporum (JPSK3), P. microspora (JPSK19), G. mangiferae (JPSK25), and ascorbic acid used as a positive control. Values are expressed as mean ± standard deviation (n = 3). Mean values do not share common superscripts and have significant statistical differences (P < 0.05).
Figure 2

Antioxidant activities of the fungal extract (a) DPPH and (b) H2O2. (c) Power reducing and (d) superoxide radical assay. C. brevisporum (JPSK3), P. microspora (JPSK19), G. mangiferae (JPSK25), and ascorbic acid used as a positive control. Values are expressed as mean ± standard deviation (n = 3). Mean values do not share common superscripts and have significant statistical differences (P < 0.05).

Table 2

IC50 values of the fungal extract

Sample code Fungal extract DPPH IC50 H2O2 IC50 Power reducing IC50 Superoxide IC50
JPSK3 Colletotrichum brevisporum 115.42 ± 7.50 116.28 ± 8.37 ND* ND*
JPSK19 Pestalotiopsis microspora 61.21 ± 2.92 63.19 ± 1.32 54.60 ± 4.39 87.93 ± 2.23
JPSK25 Guignardia mangiferae 90.41 ± 1.25 84.46 ± 2.40 83.75 ± 2.12 103.39 ± 4.09
A.A Ascorbic acid (Standard) 42.375 ± 7.17 46.91 ± 1.95 45.95 ± 4.31 33.40 ± 7.51

*ND not detectable. Data were depicted as mean ± SD (n = 3). Mean data do not share common superscripts and have significant differences (P < 0.05).

3.2.2 H2O2 scavenging assay

H2O2 is present in small amounts in air, water, plants, bacteria, and the human body. H2O2 is decomposed into oxygen and water, which might result in the formation of hydroxyl radicals (˙OH). Antioxidants can neutralize hydrogen peroxide by decomposing it into molecular oxygen and water [31]. Figure 2b shows the H2O2 scavenging capability of a fungal extract at different dosages (25–125 µg/mL) and their scavenging activity ranged from 12.58 to 84.91%. Vitamin C is utilized as a standard and its scavenging activity ranged from 36.48 to 89.94%. The fungal extract, P. microspora (JPSK19), shows stronger scavenging effects (84.91%), whereas C. brevisporum (JPSK3) and G. mangiferae (JPSK25) show moderate effects of 50.31 and 69.18%, respectively, at a concentration of 125 µg/mL. The ethyl acetate extract demonstrated considerable scavenging ability in comparison with the standard. The inhibitory concentration (IC₅₀) levels are given in Table 2. According to Khalil et al. [23], the endophytic fungi from Hibiscus sabdariffa show a reduction potential in the range of 99–93%. Yadav et al. [9] reported that Eugenia jambolana Lam associated fungi endophytes show an antioxidant reduction potential ranging from 75 to 10% while, in this work, the reduction capacity (H2O2) of fungal endophytes ranges from 84.91 to 12.58%.

3.2.3 Power-reducing antioxidant assay

The reducing power assay was used to determine the electron-donating ability of an antioxidant. The reductants present in the suspension enable the Fe3+/ferricyanide complex to be reduced to the ferrous form. As a result, Fe2+ can be determined at 700 nm. The enhanced absorbance in the reaction mixture suggested that the sample had a greater reducing power capacity. Previous studies reported that reducing abilities break the free-radical chain by donating an atom of hydrogen [32,33]. This result shows that the ethyl acetate extracts of C. brevisporum (JPSK3), P. microspora (JPSK19), and G. mangiferae (JPSK25) had absorbance values of 0.075, 0.125, and 0.111, respectively. The reducing capacity of the fungal extract was dependent on concentrations ranging from 25 to 125 μg/mL. The standard drug absorbance value was found to be 0.156 at 125 µg/mL. Among the three fungal extracts, P. microspora (JPSK19) shows increased reducing capacity when compared with the control. The reducing power of all fungal extracts and standard absorbance values are depicted in Figure 2c. The IC50 values were calculated and are given in Table 2. Similar to our study, Gunasekaran et al. [30] reported that Alternaria sp. shows higher reducing power capacity.

3.2.4 Superoxide radical scavenging assay

Superoxide is an oxygen-centred radical with a narrow range of reactivity. Superoxide has low chemical reactivity but can produce more hazardous species such as singlet oxygen and hydroxyl radicals, both of which contribute to oxidative stress [34]. In the superoxide assay, antioxidants can prevent the generation of blue NBT, which has a wavelength of 560 nm. The decrease in antioxidant absorbance indicates that O2 ions are being consumed in the reaction solutions. In this study, three fungal extracts from C. brevisporum (JPSK3), P. microspora (JPSK19), and G. mangiferae (JPSK25) were evaluated using superoxide assay in the concentration range of 25–125 µg/mL; the extracts exhibited an antioxidant activity ranging from 6.60 to 78.85%. The extract from P. microspora (JPSK19) exhibits a high activity of 78.85% at a concentration of 125 µg/mL, whereas G. mangiferae (JPSK25) has moderate effects (69.60%). When compared to P. microspore and G. mangiferae fungi, C. brevisporum (JPSK3) exhibits substantially reduced activity (40.52%). The standard drug’s scavenging effect was found to be 87.22% at 125 µg/mL, as shown in Figure 2d. The IC50 values were determined and are shown in Table 2. It has been reported [24] that the ethyl acetate extract of Colletotrichum gloeosporioides exhibited antioxidant properties with IC50 concentration of 67.46 ± 0.576 μg/mL, whereas the IC50 value of the standard was 29.51 ± 3.642 μg/mL.

3.3 Antibacterial activities

Three different fungal extracts were evaluated for their antibacterial effects against four human pathogens B. cereus, S. aureus, E. coli, and S. typhi. All the fungal extracts showed good activities against the selected human pathogenic bacterial strains. The results revealed that C. brevisporum (JPSK3), P. microspora (JPSK19), and G. mangiferae (JPSK25) showed effective antibacterial activities against all four organisms from minimum to maximum concentrations (25–125 µg/mL). Among them, P. microspora (JPSK19) was more effective against all four microorganisms, E. coli (1.40 ± 0.10), S. typhi (1.13 ± 0.30), S. aureus (1.30 ± 0.00), and B. cereus (1.00 ± 0.10), at a dosage of 125 µg/mL. However, the fungus C. brevisporum (JPSK3) was more effective against B. cereus (1.10 ± 0.10) and S. aureus (1.13 ± 0.06) but less active in E. coli (0.77 ± 0.12) and S. typhi (0.93 ± 0.15). The fungus G. mangiferae (JPSK25) was also more effective on S. aureus (1.03 ± 0.05) and B. cereus (1.00 ± 0.10) and less active in E. coli (0.86 ± 0.05) and S. typhi (0.73 ± 0.15) at a dosage of 125 µg/mL. The standard antibiotic streptomycin was employed as a standard. All the studies were conducted in triplicates and the inhibition zones were measured and are shown in Table 3.

Table 3

Antibacterial activities of the fungal crude extract compounds (zone of inhibition was measured in cm)

Antibacterial activities (zone of inhibition incm) Pathogenic microorganisms Concentration (µg/mL) Endophytic fungal crude compounds Streptomycin (+ve control) 50 µg/mL
C. brevisporum (JPSK3) P. microspore (JPSK19) G. mangiferae (JPSK25)
Gram-negative E. coli 25 0.30 ± 0.10
50 0.40 ± 0.10 0.46 ± 0.05 0.43 ± 0.05 1.20 ± 0.10
75 0.53 ± 0.15 0.63 ± 0.05 0.66 ± 0.11
100 0.60 ± 0.10 1.03 ± 0.15 0.66 ± 0.05
125 0.77 ± 0.12 1.40 ± 0.10 0.86 ± 0.05
S. typhi 25 0.46 ± 0.11
50 0.36 ± 0.11 0.96 ± 0.20 0.46 ± 0.11 1.80 ± 0.10
75 0.43 ± 0.05 0.93 ± 0.15 0.43 ± 0.11
100 0.66 ± 0.11 1.06 ± 0.15 0.66 ± 0.01
125 0.93 ± 0.15 1.13 ± 0.30 0.73 ± 0.15
Gram-positive B. cereus 25 0.30 ± 0.10
50 0.33 ± 0.05 0.53 ± 0.11 0.46 ± 0.05 1.30 ± 0.10
75 0.83 ± 0.05 0.56 ± 0.11 0.66 ± 0.05
100 1.00 ± 0.17 0.66 ± 0.25 0.86 ± 0.05
125 1.10 ± 0.10 1.00 ± 0.10 1.00 ± 0.10
S. aureus 25 0.46 ± 0.05 0.76 ± 0.11 0.50 ± 0.00
50 0.73 ± 0.05 0.96 ± 0.05 0.66 ± 0.05 1.40 ± 0.26
75 0.83 ± 0.05 1.10 ± 0.17 0.83 ± 0.05
100 0.96 ± 0.11 1.23 ± 0.05 0.96 ± 0.11
125 1.13 ± 0.06 1.30 ± 0.00 1.03 ± 0.05

3.4 Characterization of fungal bioactive compounds

3.4.1 GC-MS profiling and analysis

GC-MS is a comprehensive analytical technique that enables the identification of diverse substances present in a given sample. This technique is able to detect numerous compounds, including hydrocarbons, alcohols, phenols, esters, alkaloids, steroids, amino acids, and nitro compounds. The extracts of the selected endophytic fungi C. brevisporum (JPSK3), P. microspora (JPSK19), and G. mangiferae (JPSK25) were examined by GC-MS [35,36] to identify the bioactive compounds profile.

According to the gas chromatography results, the major compounds found in three fungal extracts are as follows. In C. brevisporum (JPSK3), 5-eicosene, (E)- (14.55%), 9-eicosene, (E)- (11.13%), phenol 4-(1,1,3,3-tetramethylbutyl (9.46%), acetamide, N-(3-methylphenyl) (9.06%), 1-docosene (7.80%), 1-n-hexyladamantane (5.96%), tricyclo[4.3.1.1(3.8)]undecane-3-carboxylic acid, methyl ester (5.23%), acetamide, N-(3-methylphenyl)- (4.22%), phenol, m-tert-butyl (3.74%), 1,2-benzenedicarboxylic acid, and butyl 2-methylpropyl ester (3.74%) were found (Table 4). In P. microspora (JPSK19), 1-octadecene (12.18%), 2,6-difluorotoluene (10.27%), phenol, p-tert-butyl- (8.65%), phenol, 4-(1,1-dimethylpropyl) (8.29%), 1-adamantanecarboxylic acid, 2-propenyl ester (8.00%), phenol, 4-(1,1,3,3-tetramethylbutyl) (5.49%), pentanoic acid, 5-hydroxy-, p-t-butylphenyl ester (5.40%), and dibutyl phthalate (4.59%) were found (Table 5). In G. mangiferae (JPSK25), 9-eicosene,(E)- (12.92%), 1-nonadecene (11.74%), 1-docosene (7.83%), 4-tert-butylphenyl acetate (6.07%), 4-tert-butylphenol (6.15%), desaspidinol (1-(2,6-dihydroxy-4-methoxyphenyl)-1-butanone) (5.35%), cyclododecane (4.83%),1-n-hexyladamantane (3.89%), phenol, 4-(1,1-dimethylpropyl) (3.79%), phenol, 2-methyl-4-(1,1,3,3-tetramethylbutyl) (3.76%), and phenol, 4-(1,1,3,3-tetramethylbutyl) (3.67%) were found (Table 6). The compounds synthesized by endophytic fungus have the potential to serve as a viable option for promoting human well-being. The utilization of the entire plant by traditional practitioners can be rationalized by the presence of several therapeutic components [37]. The active compounds with their RT molecular formulas, concentration percentages, and CAS# (Chemical Abstracts Service) are given in Table 6.

Table 4

Main compounds identified in the fungal extract of Colletotrichum brevisporum (JPSK3) by GC-MS analysis

Sl. no. RT Name of the compounds Molecular weight Molecular formula Area % CAS\#
1 11.986 Phenol, 2,4-bis(1,1-dimethylethyl) 191.20 C14H22O 0.58 000006-76-4
2 12.842 7-Hexadecene (Z) 55.10 C16H32 0.79 035507-09-6
3 13.531 Phenol, 4-(1,1-dimethylpropyl) 135.10 C11H16O 1.14 000080-46-6
4 13.808 2-Propenoic acid, tridecyl ester 69.10 C12H24 1.24 003076-04-8
5 13.931 1-n-Hexyladamantane 136.05 C14H22O 5.96 022458-75-9
6 14.008 Acetamide, N-(3-methylphenyl) 135.10 C22H28N2O 9.06 000537-92-8
7 14.086 Phenol, 4-(1,1,3,3-tetramethylbutyl 135.10 C14H22O 9.46 000140-66-9
8 14.197 Phenol, m-tert-butyl- 135.10 C10H14O 3.74 000585-34-2
9 14.375 Phenol, 4-(1,1,3,3-tetramethylbutyl)- 135.10 C22H28N2O 4.22 000537-92-8
10 14.453 Tricyclo[4.3.1.1(3,8)]undecane-3-carboxylic acid, methyl ester 149.20 C 14 H22O 6.39 000140-66-9
11 14.730 9-Eicosene (E)- 55.10 C12H18O2 5.23 031061-61-7
12 14.842 Phenol, p-tert-butyl- 70.10 C10H14O 0.30 074685-29-3
13 15.341 1,2-Benzenedicarboxylic acid, bis (1-methylpropyl) ester 149.10 C20H40 0.90 000098-54-4
14 15.564 2,4,6-Trimethylpropiophenone 147.20 C16H22O4 0.51 000605-45-8
15 15.753 1,2-Benzenedicarboxylic acid, bis (2-methylpropyl) ester 149.10 C12H16O 0.57 002040-15-5
16 15.886 Phthalic acid, isobutyl undecyl ester 149.10 C16H22O4 3.40 000084-69-5
17 16.152 1,2-Benzenedicarboxylic acid, butyl 2-methylpropyl ester 149.10 C23H36O4 0.73 1000308-97-3
18 16.297 Phthalic acid, butyl isohexyl ester 149.10 C16H22O4 3.74 017851-53-5
19 16.441 5-Eicosene, (E)- 57.20 C18H26O4 1.65 1000309-03-6
20 17.986 1-Docosene 83.20 C20H40 14.55 074685-30-6
21 19.419 1-Heneicosyl formate 69.20 C22H44 7.80 001599-67-3
22 20.352 Phthalic acid, 2-ethylhexyl isohexyl ester 71.20 C22H44O2 4.03 077899-03-7
23 20.741 Trichloroacetic acid, hexadecyl ester 97.20 C22H34O4 0.76 1000308-98-5
24 21.963 Nonadecyl trifluoroacetate 97.20 C18H33Cl3O2 1.81 074339-54-1
Table 5

Main compounds identified in the fungal extract of Pestalotiopsis microspore (JPSK19) by GC-MS analysis

Sl. No. RT Name of the compounds Molecular weight Molecular formula Area % CAS\#
1 11.831 Methyl cis-2-methyl-2-butenoate 55.10 C5H8O 2 0.60 1000143-72-2
2 11.964 Phenol, 2,4-bis(1,1-dimethylethyl)- 191.20 C14H22O 1.09 000096-76-4
3 12.830 1-Hexadecene 55.20 C16H32 0.59 000629-73-2
4 13.830 Acetamide, N-(2,6-dimethylphenyl)- 121.10 C10H13NO 2.92 002198-53-0
5 13.930 Phenol, 4-(1,1,3,3-tetramethylbutyl)- 135.20 C14H22O 5.49 000140-66-9
6 14.008 Phenol, 4-(1,1-dimethylpropyl)- 135.20 C11H16O 8.29 000080-46-6
7 14.086 Phenol, p-tert-butyl- 135.20 C10H14O 8.65 107144-95-6
8 14.186 Phenol, 4,4′-(1,2-diethyl-1,2-ethanediyl) bis- 135.20 C14H12O 2 3.52 000084-16-2
9 14.275 Acetamide, N-(3-methylphenyl)- 149.20 C22H28N2O 3.77 000537-92-8
10 14.364 Pentanoic acid, 5-hydroxy-, p-t-butylphenyl ester 136.20 C15H22O3 5.40 166273-37-6
11 14.441 2-Methyl-4-hydroxybenzoxazole 149.20 C8H7NO2 4.40 051110-60-2
12 14.730 9-Eicosene, (E)- 55.20 C20H40 6.22 074685-29-3
13 14.841 Adipic acid, 1-adamantylmethyl butyl ester 135.20 C21H34O4 0.65 1000324-38-4
14 15.063 3-Cyclopentylpropionic acid, 4-cyanophenyl ester 138.30 C8H14O2 0.47 1000307-13-4
15 15.341 1,2-Benzenedicarboxylic acid, bis-2-methylpropyl) ester 149.10 C16H22O4 0.45 000084-69-5
16 15.463 2,6-Difluorotoluene 127.10 C7H6F2 10.27 000443-84-5
17 15.563 2,4,6-Trimethylpropiophenone 127.10 C12H16O 0.80 002040-15-5
18 15.752 Dibutyl phthalate 149.10 C16H22O4 4.59 000084-74-2
19 15.886 Phthalic acid, isobutyl nonyl ester 149.10 C21H32O4 0.76 1000309-04-4
20 16.152 1,2-Benzenedicarboxylic acid, butyl oxtyl ester 149.10 C20H30O4 3.29 000084-78-6
21 16.286 1,2-Benzenedicarboxylic acid, butyl 2- ethylhexyl ester 149.10 C20H30O4 1.90 000085-69-8
22 16.430 1-Octadecene 55.20 C18H36 12.18 000112-88-9
23 16.919 2H-1,3-Benzimidazol-2-one, 5-amino-1, 3-dihydro- 149.10 C7H7N 3 O 0.75 1000338-09-4
24 17.985 E-14-Hexadecenal 97.20 C16H30O 4.86 330207-53-9
25 18.952 3,7-Dimethyl-1,1-bis(trimethylsilyl) octa-2,6-diene 125.10 C10H18 1.81 1000193-82-5
26 19.418 1-Docosene 55.20 C22H44 2.83 001599-67-3
27 20.352 1,2-Benzenedicarboxylic acid, diisooctyl ester 149.10 C34H58O4 0.87 027554-26-3
28 20.741 n-Tetracosanol-1 83.20 C24H50O 1.50 000506-51-4
29 21.963 2-Pentadecanol 207.10 C15H32O 0.62 001653-34-5
Table 6

Main compounds identified in the fungal extract of Guignardia mangiferae (JPSK25) by GC-MS analysis

Sl. No. RT Name of the compounds Molecular weight Molecular formula Area % CAS\#
1 11.975 Phenol, 2,4-bis(1,1-dimethylethyl)- 191.20 C14H22O 1.04 000096-76-4
2 12.842 9-Octadecene, (E) 83.20 C18H36 1.03 007206-25-9
3 12.919 Cyclooctacosane 57.20 C28H56 0.57 000297-24-5
4 13.530 Phenol, 4,4′-(1,2-diethyl-1,2-ethanediyl) bis- 135.20 C18H22O2 0.91 000084-16-2
5 13.808 Cyclododecane 55.10 C12H24 4.83 000294-62-2
6 13.930 1-n-Hexyladamantane 136.20 C16H28 3.89 022458-75-9
7 14.008 Phenol, p-tert-butyl- 135.20 C10H14O 6.15 000098-54-4
8 14.086 4-tert-Butylphenyl acetate 135.20 C12H16O2 6.07 003056-64-2
9 14.197 Phenol, 4-(1,1-dimethylpropyl)- 135.20 C11H16O 3.79 000080-46-6
10 14.275 1-(6-Methyl-2-pyridyl)propan-2-one 149.20 C9H11NO 2.54 065702-08-1
11 14.375 Phenol, 4-(1,1,3,3-tetramethylbutyl)- 135.20 C14H22O 3.67 000140-66-9
12 14.441 Phenol, 2-methyl-4-(1,1,3,3-tetramethylbutyl)- 149.20 C15H24O 3.76 002219-84-3
13 14.664 5,6-Dimethoxy-1-indanone 192.20 C11H12O3 2.30 002107-69-9
14 14.730 1-Nonadecene 55.10 C19H38 11.74 018435-45-5
15 14.841 2,6-Nonadienal, (E,E)- 70.10 C9H14O 1.03 017587-33-6
16 15.319 Desaspidinol 167.20 C11H14O4 5.35 000437-72-9
17 15.752 1,2-Benzenedicarboxylic acid, bis 2-methylpropyl) ester 149.10 C16H22O4 2.64 000084-69-5
18 15.886 1,2-Benzenedicarboxylic acid, butyl octyl ester 149.10 C20H30O4 0.53 000084-78-6
19 16.152 Phthalic acid, cycloheptyl isobutyl ester 57.20 C18H24O4 2.39 1000322-82-8
20 16.297 1,2-Benzenedicarboxylic acid, butyl cyclohexyl ester 149.10 C18H24O4 1.20 000084-64-0
21 16.430 9-Eicosene, (E)- 55.20 C20H40 12.92 074685-29-3
22 16.575 Naphthalene, 1-phenoxy- 220.20 C16H12O 1.29 003402-76-4
23 16.641 Octadecane, 1-(ethenyloxy)- 57.20 C20H40O 0.97 000930-02-9
24 17.330 Thiourea, N-(1-methylpropyl)-N′-phenyl- 208.20 C11H16N2S 1.91 015093-37-5
25 17.985 1-Docosene 83.20 C22H44 7.83 001599-67-3
26 19.419 3-Eicosene, (E)- 57.20 C20H40 4.62 074685-33-9
27 20.352 Di-n-octyl phthalate 149.10 C24H38O4 0.75 000117-84-0
28 20.741 2-Chloropropionic acid, octadecylester 57.20 C3H5ClO2 2.57 088104-31-8
29 21.963 Pentadecyl trifluoroacetate 207.10 C17H31F3O2 1.18 1000351-74-4
30 23.340 Tetrasiloxane, decamethyl- 207.20 C10H30O3Si4 0.54 000141-62-8

In the current study, the chemical groups were also examined. The following chemical groups (phenolic, fatty acid, terpenoids, alkaloids, and poliketides) were discovered. The phenol group was more dominant in all three fungi, C. brevisporum (54%), P. microspora (66%), and G. mangiferae (35%), as shown in Figure 3a. Among all compounds in P. microspora (JPSK19), p-tert-butylphenol was selected because of its more potential activities such as antibacterial, antioxidant, and anticancer [38]. Therefore, p-tert-butylphenol was considered for further studies of compound characterization by spectral and analytical analysis by comparing with the commercially available standard compound p-tert-butylphenol (TCM Company).

Figure 3 
                     GC-MS chemical profiles of the fungal extract: (a1) C. brevisporum (JPSK3), (a2) P. microspora (JPSK19), and (a3) G. mangiferae (JPSK25). (b) TLC plate No. 1 analysis of the fungal extract (spot 1 – JPSK3, 2 – JPSK25, 3 – JPSK19, and 4 – standard) while TLC Plate No. 2 analysis of the fungal eluted fraction(spot 3 – JPSK3 and spot 4– standard). (c) GC-MS analysis of TLC eluted fractions (1) and standard (2). (d) TLC bioautography methods of antibacterial activities (1 – S. typhi, 2 – B. cereus, 3 – E. coli, and 4 – S. aureus).
Figure 3

GC-MS chemical profiles of the fungal extract: (a1) C. brevisporum (JPSK3), (a2) P. microspora (JPSK19), and (a3) G. mangiferae (JPSK25). (b) TLC plate No. 1 analysis of the fungal extract (spot 1 – JPSK3, 2 – JPSK25, 3 – JPSK19, and 4 – standard) while TLC Plate No. 2 analysis of the fungal eluted fraction(spot 3 – JPSK3 and spot 4– standard). (c) GC-MS analysis of TLC eluted fractions (1) and standard (2). (d) TLC bioautography methods of antibacterial activities (1 – S. typhi, 2 – B. cereus, 3 – E. coli, and 4 – S. aureus).

3.4.2 TLC profiling and analysis

TLC profiling of three different fungal extracts of compounds provides valuable outcomes that led to the discovery of various biochemical compounds. The R f values obtained from the fungal extract provide pivotal data about their polarity and important clues to isolate these compounds in the separation process. Different R f values of the compounds also reflect on their polarities of the various solvent systems for TLC studies and the selection of the appropriate solvent system. This knowledge will aid in the selection of a suitable solvent system for subsequent chemical separation from these fungal extracts. The TLC study of the fungal extracts shows many visible fractions on the plate having different R f values. The presence of bioactive compounds was confirmed on TLC silica plates when observed under UV at 366 nm. All fungal fractions were observed and their R f values were calculated. The sample spot 1 (JPSK3) shows three fractions (R f = 0.07, 0.32, and 0.57), spot 2 (JPSK25) shows two fractions (R f = 0.06 and 0.59), spot 3 (JPSK19) shows three fractions (R f = 0.09, 0.32, and 0.61), and the R f value of spot 4 (Standard) was 0.64. Among all the three fungal extracts, the spot 3 (JPSK19) sample showed a very close R f value towards the standard compound, as shown in Figure 3b.

Further eluted fraction of the fungal extract (JPSK19) was reconfirmed by GC-MS, and a similar range of peak was observed, as shown in Figure 3c. The biological potential (antibacterial activities) of the similar fraction was confirmed by TLC bioautography against four human pathogens; the fractions showed good antibacterial activities as shown in Figure 3d. Further, the compositional and structural data of the compound and functional groups [39] were analysed by UV, FT-IR, HPLC, and NMR.

3.4.3 UV-vis spectrophotometer analysis

The partially purified fungal compound using the TLC technique was investigated by UV-visible spectrometry. The high absorbance was observed at 273 nm. The UV-vis spectroscopy results show that the absorbance rate of the fungal compound was associated with the standard compound (p-tert-butylphenol), as shown in Figure 4a.

Figure 4 
                     UV absorption spectrum of the fungal extract and standard: (a) FT-IR spectrum analysis (1 – fungal extract 2 – standard). (b) HPLC analysis of the fungal extract: (1) red arrow fungal extract and (2) standard (c).
Figure 4

UV absorption spectrum of the fungal extract and standard: (a) FT-IR spectrum analysis (1 – fungal extract 2 – standard). (b) HPLC analysis of the fungal extract: (1) red arrow fungal extract and (2) standard (c).

3.4.4 FT-IR spectroscopy analysis

The FT-IR spectra show vibrations that proved the data on the chemical nature of the partially purified fungal compound by comparison with the commercially available standard. The bands in the IR spectrum persuasively depicted the similar chemical nature of the fungal extracted compound (p-tert-butylphenol) with that of the commercially available standard compound. The hydroxyl group of the phenol was absorbed strongly in the 3,500–3,000 cm−1 region. In the FT-IR spectra, our compound showed a strong absorption band at 3300.04 cm−1 and was assigned to the OH stretching vibration, indicating the presence of a hydroxyl group (–OH). The CH stretching vibrations of aromatic structures are predicted to appear in the 3,100–3,000 cm−1 frequency ranges, with several weak bands. The CH in plane vibrations showed sharp but weak-to-moderate intensity bands in the 1,500–1,000 cm−1 region. The FT-IR spectra of the aromatic CH stretching vibrations of our sample show weak to medium bands at 1486.82 cm−1, indicating the presence of a CH group. The stretching vibration of the aromatic ring C═C occurs in the region 2,000–1,500 cm−1. The FT-IR spectrum of the C═C stretching vibration of our compound was found in the 1585.85 cm−1 region. The C–O stretching vibrations in phenols are thought to exhibit the strongest band in the 1,300–1,200 cm−1 range, and the major peak stretching vibration was observed for our compound at 1199.65 cm−1 and was assigned to the strong mode of C–OH. The symmetric CH3 bending area of the IR spectra is characterized by the tertiary butyl group giving rise to CH bending modes. The major peak, which indicates the presence of p-tert butyl phenol, was found at 1365.13 cm−1 similar to those of previous studies [40,41]. Based on the stretching vibrations, the presence of p-tert-butylphenol, a phenol compound was obtained in our sample. Furthermore, the entire peak observed in our extracted sample was similar to the commercially available standard compound, as shown in Figure 4b.

The FT-IR spectra validate that functional groups are present in the partially purified fungal compound and helped to identify and evaluate the functional groups. The outcomes of this work corroborate with an earlier work that reported numerous fungal compounds [41]. Several works employed FT-IR spectra as a tool for characterizing the bioactive compounds of plants and nanomedicines [42,43]. The results in this work emphasize that the compounds were developed from endophytic fungi of ethnomedicinal plants.

3.4.5 HPLC analysis

The fungal extract of P. microspora was characterized and analysed with the commercial standard compound by HPLC. Based on this analysis, the partially purified fungal compound has a prominent peak with an RT of 1.912 min, and the RT of the standard compound was 1.915 min at an absorption of 243 nm. The HPLC analysis revealed that the fungal and standard compound and their RTs were comparable and were close [41,44]. Therefore, both the partially purified fungal and standard compounds in the recorded chromatograms suggest the possibility that they are the same compounds (Figure 4c).

3.4.6 NMR analysis

Further confirmation of the fraction of TLC eluted analysis was done by UV vis spectrophotometry, HPLC, FT-IR, and NMR analysis with the standard. We further investigated the structural differences of the compound from the eluted fraction of our sample (JPSK19) and the p-tert-butylphenol by 1H and 13C NMR. The spectrogram taken with 1H NMR (400 MHz, CdCl3) is shown in Figure 5. Analysis of the selected sample (JPSK19) was evaluated and chemical shifts include: δ 6.96 ppm (1 OH) (d, 7.82 Hz, 1H), 7.17 (4, 5 CH) (t, J = 7.85 Hz, 1H), 6.67 (7, 8 CH) (t, J = 7.85 Hz, 1 H), 1.29 (9, 10, 11 CH3) (s, J = 7.85 Hz, 1H) for the molecular formula (C10H14O). It is evident from 1H NMR spectra (Figure 5a1) that three singlets at δ = 1.29 are closer to carbon-3 (δ = 34.69 ppm) and two singlets at δ = 7.17 are found. Out of the two, one is closer to carbon-2 (δ = 153.46 ppm) and carbon number-7 (δ = 112.44 ppm), while the other singlet is closer to carbon-8 (δ = 112.11 ppm) and also closer to carbon-2 (δ = 153.46 ppm). The two singlets at δ = 6.67 are found and out of the two, one is closer to carbon-4 (δ = 129.27 ppm) and carbon-6 (δ = 155.15 ppm) and the other singlet is closer to carbon-5 (δ = 129.27 ppm) and carbon-6 (δ =155.15), whereas one OH (δ = 6.96 ppm) is away from carbon-3 (δ = 34.69 ppm) in the same ring. The aromatic protons are para-coupled to three singlets (1.29), which are closer to carbon-3 (δ = 34.69 ppm); thus, this indicates the presence of tert-butyl moiety at the para position, one aromatic ring moiety, and one OH moiety in the eluted fraction of the sample JPSK19. The spectrogram obtained with 13C NMR (400 MHz, CdCl3) is shown in Figure 5. The analysis of the selected sample (JPSK19) of the fraction was evaluated and it is evident from the 13C NMR spectrum shown in Figure 5b1 that three carbons at δ = 31.30 having three singlets at 1.29 (1H spectrum) are closer to carbon-3 (δ = 34.69 ppm); one carbon at δ = 34.69 are found closer to carbon-2 (δ = 153.46 ppm) and carbon-2 (δ = 153.46 ppm) and are closer to carbon-4 and -5 (δ =129.27 ppm), having two singlets at δ = 6.67, respectively. However, two carbons at δ = 112.44 ppm were found closer to carbon having δ = 155.15 ppm, whereas out of the two carbons, one carbon (δ = 112.44 ppm) was closer to carbon at δ = 129.27 ppm and other carbon (δ = 112.44) was close to carbon at δ = 129.27 ppm. Thus, this suggested that these carbons (C at δ = 153.46 ppm, two C at δ = 129.27 ppm, two C at δ = 112.44, and one carbon at δ = 155.15 ppm are attached and form aromatic rings, in which the para position carbon at 153.46 ppm is attached to carbon (δ 34.69 ppm), having three singlets at 1.29 (1H spectrum), whereas the carbon at δ = 155.15 ppm is closer to δ = 6.96 ppm (1 OH) (d, 7.82 Hz, 1H) [45,46,47].

Figure 5 
                     NMR spectral analysis. (a) 1H NMR (1 – fungal extract, 2 – prediction structure, and 3 – standard). (b) 13C NMR (1 – fungal extract, 2 – prediction structure, and 3 – standard). (c) Structure of p-tert-butylphenol.
Figure 5

NMR spectral analysis. (a) 1H NMR (1 – fungal extract, 2 – prediction structure, and 3 – standard). (b) 13C NMR (1 – fungal extract, 2 – prediction structure, and 3 – standard). (c) Structure of p-tert-butylphenol.

The chemical shift values of the protons in the spectrum precisely corresponded to their corresponding functional groups. The structure explicated from 1H and 13C NMR spectrum by Mestrenova (C10H14O) was matched with the spectra and structure of p-tert-butylphenol, respectively, which was noted from the PubChem compound. The compound structure explicated from the spectrogram of the JPSK19 sample (eluted fraction of TLC) and the Pub Chem was identical to p-tert-butylphenol. This study verifies the occurrence of p-tert-butylphenol in the eluted fraction of the sample JPSK19 of P. microspora.

A further investigation was performed to study the influence of p-tert-butylphenol on anticancer activities under in vitro and in vivo conditions. The present work aimed to specifically demonstrate compound characterization of partially purified fungal compounds. The RT for the major compound identified from HPLC was then studied by 1H NMR for structural configuration. FT-IR spectroscopy analysis was used to identify the compositional data functional groups of the compound [39].

NMR relies on the detection of resonance signals emitted by rotating atomic nuclei within a magnetic field. This technique is particularly valuable in several biological investigations due to its ability to examine naturally existing isotopes, such as 1H and 13C. The location of a resonance signal within an NMR spectrum is dependent upon the chemical environment around the specific atom being analysed. These positions are denoted as chemical shifts, which are expressed relative to a reference molecule. The magnitude of a signal peak is directly related to the quantity of atoms present; therefore, it is feasible to measure the concentration of metabolites based on the resonance signal [48]. The use of 1H NMR spectroscopy enables the acquisition of substantial quantities of data pertaining to the composition or dynamics of metabolites inside cellular constituents. The challenges associated with the presence of a high-intensity resonant solvent or residual water can be effectively addressed by using specialized pulse sequences. These sequences are designed to mitigate the interference caused by these substantial signals, thereby ensuring that the pertinent information within the same shift region remains discernible. In contrast, solid or semi-solid materials exhibit broader line widths and spectral overlap as a result of isotropic interactions. NMR spectra were employed as input data in a multivariate principal component analysis to discern the chemical makeup of the isolated compounds from the biological sources.

4 Conclusion

This study concludes the existence of bioactive natural compounds in the endophytic fungal extract and their antioxidant and antibacterial effects. According to the findings of the study, a partially extracted fungal compound isolated from the ethno-medicinal plant Bergenia ciliata has potential novel medicinal properties. This study reports on endophytic fungi that may support the development of novel lifesaving drugs in the future, and it concludes that the effective use of keystone species may protect human life. The main limitation of this work is that we failed to isolate and characterize the pure bioactive compound from the endophytic fungi of Bergenia ciliata. Further studies are warranted to isolate and evaluate the bioactive compound in vitro and in vivo for their therapeutic use in cancer treatment.



Acknowledgements

The authors would like to thank the instrumentation facilities provided by DST-PURSE, RUSA, and the Department of Molecular Microbiology, School of Biotechnology, Madurai Kamaraj University in Madurai, Tamil Nadu. The author wishes to thank the Government of Sikkim, India, for providing a fellowship (Post Metric Scholarship Scheme) in exchange for their assistance and Princess Nourah bint Abdulrahman University Researchers Supporting Project number (PNURSP2023R357), Princess Nourah bint Abdulrahman University, Riyadh, Saudi Arabia.

  1. Funding information: This research was supported by Princess Nourah bint Abdulrahman University Researchers Supporting Project number (PNURSP2023R357), Princess Nourah bint Abdulrahman University, Riyadh, Saudi Arabia.

  2. Author contributions: M.P.: conceptualization, supervision, and validation; S.S. and A.S.A.: data curation, methodology, and writing – original draft; W.A. and C.G.: formal analysis and validation; J.R.P.: resources; P.R. and N.R.: validation and writing – review & editing; A.S.A.: validation and formal analysis.

  3. Conflict of interest: The authors declare no conflict of interest.

  4. Ethical approval: The authors conducted their research with no animal or human participants.

  5. Data availability statement: The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

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Received: 2023-09-04
Revised: 2023-10-19
Accepted: 2023-10-30
Published Online: 2023-11-21

© 2023 the author(s), published by De Gruyter

This work is licensed under the Creative Commons Attribution 4.0 International License.

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