UMYU Scientifica

A periodical of the Faculty of Natural and Applied Sciences, UMYU, Katsina

ISSN: 2955 – 1145 (print); 2955 – 1153 (online)

ORIGINAL RESEARCH ARTICLE

Characterization and Antibacterial Activity of Endophytic Aspergillus niger strain MOR-1 Isolated from Moringa oleifera

Abdullahi Ibrahim Dalhatu¹, Bahauddeen Salisu²٭, Mukhtar Gambo Lawal², and Hayatudeen Muhammad Rumah²

¹Department of Microbiology, College of Natural and Applied Sciences, Al-Qalam University, PMB 2137, Katsina, Nigeria

²Department of Microbiology, Faculty of Science, Umaru Musa Yar'adua University, PMB 2218, Katsina, Nigeria

٭Corresponding Author: Baha’uddeen Salisu bahauddeen.salisu@umyu.edu.ng

Abstract

The increase in multidrug-resistant (MDR) microorganisms has driven the global search for new antimicrobial drugs from untapped natural sources. One of the biggest sources of bioactive secondary metabolites is endophytic fungi, symbiotic microorganisms that inhabit plant tissues. It is true that, despite the abundance of medicinal plant species in the state of Katsina, Nigeria, there is no scientific knowledge of the antibacterial potential of the endophytic fungi of these plants. Thus, this paper seeks to isolate and profile endophytic fungi towards new antibacterial derivatives. This study isolated Aspergillus niger from Moringa oleifera, and the exometabolites were extracted, and their antibacterial activity against 10 pathogenic bacterial strains was measured using the disc diffusion technique. The fungal extract was analyzed through Gas Chromatography-Mass Spectrometry (GC-MS), Fourier Transform Infrared Spectroscopy (FTIR), in silico molecular docking to estimate binding affinities with bacterial enzymes (ECPR, MurA, and PBPs), and Adsorption, Distribution, Metabolism, Excretion/Toxicity (ADME/T) to establish the drug likeness. The antibacterial assay showed that the Aspergillus niger strain MOR-1 exhibited broad and strong activity, with inhibitory diameters ranging from 16.67 ± 1.53 mm to 29.33 ± 2.52 mm against S. aureus, P. aeruginosa, K. pneumoniae, S. pneumoniae, S. typhi, and E. coli. The activity of the strain was similar to that of ciprofloxacin (12.10 ± 1.05 to 30.1 ± 0.41 mm), indicating the synthesis of effective antibacterial metabolites with broad-spectrum activity. Analysis of the fungal extracts by GC-MS revealed various bioactive compounds, including fatty acid esters, alcohols, phenols, and aldehydes with known antimicrobial properties. The presence of functional groups (including hydroxyl, carbonyl, and alkene groups) which are typical of bioactive secondary metabolites was further confirmed by FTIR spectroscopy. The molecular docking experiments indicated that the important compounds, especially methyl ricinoleate, 9-octadecenoic acid, and methyl linolenate, exhibit high binding affinity to essential bacterial target enzymes involved in cell wall and protein synthesis. The predicted pharmacokinetic and low-toxicity profiles of the lead compounds, based on ADME/T screening, supported their potential as drug-like antibacterial candidates. The results show that Aspergillus niger is a promising source of potent antibacterial metabolites with activities similar to those of standard antibiotics. Future research should focus on purifying and testing the most promising compounds in vivo to establish their therapeutic potential as new antibacterial agents.

Keywords: Bacteria, GCMS, FTIR, Molecular docking, Antibacterial activity

INTRODUCTION

In recent years, there has been growing interest in fungal endophytes, a highly diverse group of microorganisms that inhabit plant tissues without causing visible harm to their host plants (Rai et al., 2021). First described by Anton de Bary in 1866, endophytic fungi are now recognized as ubiquitous components of plant microbiomes, occurring in nearly all plant species across diverse climatic and ecological zones, including terrestrial and marine environments (Chakraborty et al., 2021; Abdelaziz et al., 2025). Although estimates may be exaggerated, it has been suggested that approximately one million endophytic fungal species exist globally, with particularly high diversity reported in tropical and subtropical regions (Bala et al., 2025). Their occurrence and distribution are influenced by host plant species, geographical location, seasonal variation, and anthropogenic activities (Eo et al., 2022). While members of Basidiomycota and Zygomycota are present, the majority of endophytic fungi belong to the phylum Ascomycota (Lin et al., 2023).

Endophytic fungi display remarkable ecological and functional versatility, existing as mutualists, latent pathogens, or former pathogens that have lost their virulence depending on host physiology and environmental conditions (Mishra et al., 2021). This adaptability complicates their precise definition, particularly for unculturable species whose biological roles within host tissues remain unclear (Mattoo and Nonzom, 2021). Advances in metabolomics and next-generation sequencing have significantly improved the exploration of endophytic fungal diversity and biosynthetic potential, revealing these organisms as promising reservoirs of agriculturally and pharmaceutically important bioactive compounds (Bielecka et al., 2022).

The renewed scientific interest in endophytic fungi is closely linked to the escalating global challenge of antimicrobial resistance (AMR), which threatens the continued effectiveness of conventional antibiotics (Tiwari et al., 2025). The World Health Organization has identified AMR as one of the most critical public health threats of the twenty-first century, with resistant infections affecting millions of people annually and causing tens of thousands of deaths worldwide (Salam et al., 2023). This alarming trend underscores the urgent need for innovative, effective, and low-toxicity antimicrobial agents, particularly those with novel mechanisms of action and reduced potential for resistance development (Elshobary et al., 2025).

Secondary metabolites are structurally diverse, low-molecular-weight compounds produced by many organisms, particularly microorganisms, and they have historically played a central role in drug discovery (Buddhika and Abeysinghe, 2020). While plants have long been recognized as valuable sources of therapeutic agents (Dar et al., 2023), microorganisms have gained prominence due to their vast biodiversity, rapid growth, and amenability to genetic and metabolic manipulation (Thiele-Bruhn, 2021). However, extensive exploration of soil-derived microorganisms has led to frequent rediscovery of known compounds, necessitating a shift toward alternative, underexplored microbial niches (MacNair et al., 2023). In this regard, plant-associated microorganisms, particularly endophytic fungi, have emerged as promising alternative sources of chemically novel and biologically active secondary metabolites (Gakuubi et al., 2021).

Endophytic fungi occupy a unique ecological niche within plant tissues, enabling intimate interactions with host metabolic pathways and facilitating the production of structurally diverse and novel compounds not commonly synthesized by free-living microorganisms (Chandra and Enespa, 2019). This shift in natural product research emphasizes the importance of previously overlooked ecological systems in the search for new antimicrobial agents (Aghdam and Brown, 2021). Due to their adaptive metabolic plasticity, endophytic fungi can produce a broad spectrum of secondary metabolites under varying physiological and environmental conditions, particularly when subjected to co-cultivation, stress induction, or optimized culture conditions (Wadhwa et al., 2024).

The pharmaceutical relevance of endophytic fungi was notably highlighted by the discovery of Taxol production by Taxomyces andreanae, which transformed bioprospecting strategies and demonstrated the ability of endophytes to synthesize compounds previously believed to be plant-specific (Tripathi and Agnihotri, 2024). Beyond their medicinal value, endophytic fungi contribute significantly to host plant fitness by enhancing resistance to biotic and abiotic stresses such as drought, salinity, oxidative stress, heavy metal toxicity, and pathogen invasion (Verma et al., 2022; Grabka et al., 2022). These protective attributes further support their potential roles in sustainable agriculture and resistance management strategies (Tiwari and Bae, 2022).

Several classes of endophyte-derived metabolites, including alkaloids, polyketides, terpenoids, peptides, phenolics, and aliphatic compounds, have demonstrated potent antibacterial activity against a wide range of human pathogens (Das et al., 2024). Some endophytic fungal strains have shown activity against multidrug-resistant pathogens such as Pseudomonas aeruginosa, Escherichia coli, and Staphylococcus aureus, positioning them as important candidates in the global fight against AMR (Eshboev et al., 2024). Despite this potential, research on endophytic fungi remains relatively limited due to challenges in cultivation, metabolite isolation, and structural characterization (Adeleke and Babalola, 2021; Gakuubi et al., 2021). Nonetheless, the integration of omics-based approaches, including genomics, transcriptomics, proteomics, and metabolomics, continues to reveal silent biosynthetic gene clusters with significant antibacterial potential (Hur et al., 2023; Saini et al., 2024).

Although several medicinal plants in Nigeria, including those from Katsina State, have been investigated for their antibacterial properties, comparatively little attention has been given to the endophytic fungi associated with these plants. Considering the ecological diversity of Katsina State and the medicinal relevance of its indigenous flora, endophytic fungi from this region represent an underexplored but promising source of novel antibacterial compounds.

Therefore, this study aims to isolate, characterize, and evaluate Aspergillus niger associated with Moringa oleifera from Katsina State, Nigeria, for the production of novel antibacterial compounds. The findings are expected to contribute to antimicrobial drug discovery and expand current knowledge on the taxonomic and ecological diversity of endophytic fungi in this region.

MATERIALS AND METHODS

Study Area

The study took place in Katsina State, in the north of Nigeria. The area of Katsina State is 23,938 square kilometres, and its latitude and longitude are 11°08'N 13°22'N and 6°52'E 9°20'E (Ibrahim et al., 2022). The state consists of 34 Local Government Areas; it is bordered to the north by the Niger Republic; to the east by the Jigawa and Kano States; to the south by the Kaduna State; and to the west by the Zamfara State (Kankara and Abdullahi, 2024).

Collection of Plant Materials

Fresh leaves of Moringa oleifera were collected at the Biological Garden of Al-Qalam University, Katsina. After the process outlined by Shehu (2023), the plant leaf samples were packed into a plastic container and immediately transported to the laboratory. The samples were washed with warm water.

Isolation of Fungi

The plant leaves were first washed with sterile distilled water to remove any undesired dirt, as per the process employed by Qadri et al. (2013) and Zihad et al. (2022). They were then surface-sterilized with 75% ethanol, 0.5% sodium hypochlorite, and 75% ethanol, each for 1 and 3 minutes and 15 seconds, respectively, after proper washing with sterile distilled water. Then they were cut into minute pieces, about 0.5 cm2 in size, using a sharp, sterile blade, and placed on Petri dishes containing Potato Dextrose Agar (PDA) medium in a horizontal manner. 100 µg/mL streptomycin was added to the PDA medium to stop bacterial growth. Fungal colonies were cultured for 7 days in Petri dishes at room temperature. Colonies were sorted into groups based on their appearance, morphology, and color. A colony from the first culture plate was unique, so a small piece was transferred to form a pure culture. This was then spotted on Petri dishes containing PDA media that was supplemented with streptomycin. The incubation period of the plates was seven days.

Preparation of Crude Extract of Endophytic Fungal Isolate

The fungal extract was prepared using the method outlined by Zihad et al. (2022). The fungi in the pure culture plates were transferred to PDA slants and incubated for 48 hours at 30 °C to determine the growth stage of each fungus. The increasing size of each fungus was then transferred onto PDA slants in conical flasks containing 200ml autoclaved potato dextrose broth media at 121 °C for 15 minutes. The 10 days of culture in the flasks were conducted at 28 °C . The mycelial biomass was filtered using a Whatman No. 1 filter, followed by three withdrawals of the filtrate with an equal volume of ethyl acetate using a separating funnel. The extracts were pooled, and the mixture was rotary-evaporated at 400 °C to yield a crude, semisolid extract, after which it was dried into powder using a hot air ovum at 40 ⁰C. The powdered extracts were kept in vial until needed for further analysis

Evaluation of Antibacterial Activity of Crude Extract of Endophytic Fungal Isolate

Ten (10) bacterial isolates from the culture collection of the Microbiology Department of Al-Qalam University, Katsina; Umaru Musa Yar'adua University, Katsina; and Federal Teaching Hospital, Katsina were used for antibacterial screening. The following were collected: Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae, Methicillin-Resistant Staphylococcus aureus (MRSA), Streptococcus pneumoniae, Salmonella typhi, Shigella sp., Citrobacter sp. and Escherichia coli based on the list of antibiotic-resistant pathogens developed by the World Health Organization (WHO) that highlights the necessity of new antimicrobial drugs against high-risk and clinically important bacteria. The bacteria were grown in nutrient broth for 18 hours at 37 °C, after which the bacterial pellets were suspended in 5 mL of sterile normal saline and then centrifuged at 4000 rpm. The bacterial concentration was subsequently determined using the McFarland standard and diluted to an operational concentration of 5×10⁶ cfu/mL.

The disc diffusion method was employed to test the antibacterial activity of crude endophytic fungal extracts (Lykholat et al., 2021). A bacterial suspension (10⁹ cfu) was placed on Petri dishes containing Mueller-Hinton Agar (MHA). The crude extract (100 µg) was placed on sterile 6 mm diameter paper discs on the agar. The plates were incubated for 24 hours at 37 °C. The positive control (5 µg per disc) was ciprofloxacin. Antibacterial activity of the extracts was assessed by measuring the diameter (mm) of the inhibition zones they formed. The antibacterial assay was used as a preliminary screening tool to select the most potent endophytic fungal isolate for subsequent identification and chemical characterization.

Identification of the Most Potent Endophytic Fungal Isolate

The identification of the most potent fungal isolate was carried out using an integrated procedure that combined cultural and microscopic analyses, as recommended by Abdelgawad et al. (2022), Zihad et al. (2022), and Salisu et al. (2024). Three days of culture growth were carried out on PDA plates at 25 °C. To initiate the morphological identification process, glass slides and cover slips were cleaned with 95% ethanol. The microscope slide was then sanitized, and a drop of lactophenol-cotton blue was added. A dye culture plate was prepared by adding a small piece of mycelium with a needle, then a cover slip was placed on the mycelium. The slide was observed under a light microscope using a 40x objective lens, and photographs were taken.

Molecular Characterization of the Most Potent Endophytic Fungal Isolate

Isolation of DNA

The Cetyltrimethylammonium bromide (CTAB) procedure described by Singh et al. (2020) was used to extract DNA from the fungal isolates. Mortars and pestles were pre-chilled to 4 °C, and 1 g of the sample was placed in absolute alcohol for 30 minutes, after which the alcohol was left to evaporate. Then, 5 grams of the mycelium were crushed into a fine powder using a sterile mortar and pestle, and 10 mL of pre-warmed isolation buffer was combined with the crushed mycelium and stirred. Upon incubation, the mixture was allowed to cool to room temperature then 10 mL of the chloroform-isoamyl alcohol (24:1) was added and thoroughly mixed. The mixture was centrifuged at 10,000 rpm for 24 °C, after which the supernatant was transferred to a fresh sterile tube. Ice-cold isopropanol (0.6 volume) and 3 M sodium acetate (0.1 volume) were added to the aqueous layer, and the mixture was allowed to stand at -20 °C for 30 minutes. The mixture was centrifuged at 10,000 rpm for 10 minutes at 4⁰C and the supernatant was discarded. The obtained DNA pellet was washed with 75% ethanol and centrifuged once again at 10000rpm for 10 minutes at 4 °C. The ethanol was decanted, and the DNA pellet was driedine air and suspended in 200 ul of distilled water.

Polymerase Chain Reaction (PCR) Amplification

Amplification of the ITS region of the rRNA operon was performed using the forward primer (ITS-1F), which is 5'-TCCGTAGGTGAACCTGCGG-3' and the reverse primer (ITS-4R), 5'-TCCTCCGCTTATTGATATGC-3' (Salisu et al., 2024a). PCR amplification was performed at the Centre of Biotechnology, Bayero University Kano, using standard molecular biology procedures. Ration of each reaction mixture was 10 ul of Red Taq ready mix, 0.5 ul of each primer, 8 ul of sterile analytical-grade water (Sigma-Aldrich), and 5 ul of genomic DNA, 24 ul. The conditions used for thermocycling were denaturations at 94 °C for 5 min, followed by 30 cycles of denaturations at 94 °C for 1 min, annealing at 60 °C for 1 min, extension at 72 °C for 1 min, and finally an extension at 72 °C for 5 min. PCR products were separated on a 2% agarose contained on a Tris-acetate-EDTA buffer, stained with ethidium bromide, and visualized under UV light (Etanke et al., 2021). The molecular weight marker used in estimating the amplicon sizes was a 1 kb Plus DNA ladder.

DNA Sequencing and BLASTs Analysis

The amplified samples were transported to South African Medical Research Council (SAMRC) Genomic Centre for sequencing. The received sequence data were processed using the BLAST (Basic Local Alignment Search Tool) algorithm (http://blast.ncbi.nlm.nih.gov) in the NCBI database for sequence alignment and identification.

Phylogenetic Relationship

The DNA sequences obtained during this study were analyzed to construct a phylogenetic tree using the neighbour-joining method in MEGA 12 (build: 12251022-x86_64; version 12.0.15).

Gas Chromatography Mass Spectrometry Analysis

The chemical structure of fungal extracts was determined using a gas chromatography-mass spectrometry (GC-MS) system (Shimadzu GC-MS-QP2010 Plus, Japan). The instrument had an AOC-20i autoinjector set to split injection mode. Before analysis, the syringe was washed 4 times with pre-solvent and post-solvent, and 3 times with the sample, to avoid contamination. The temperature of the injection port was kept at 200-250 °C^, and the injection volume was 1 uL. The carrier was helium, operated in linear velocity flow control mode with a column flow rate of 1.8 mL/min, a total flow of about 40.8 mL/min, and a split ratio of 20:1.

The temperature of the column oven was adjusted to 70 °C and scheduled to increase to 280 °C at a rate of 10 °C/min and a hold time of 5 minutes. The interface temperature was kept at 250 °C, and the source ion temperature was 200 °C. The solvent cut time was set to 2.5, and the detector gain was set to relative mode with a threshold of 2000. Mass spectral data were recorded in scan mode, over the mass range, m/z 30-700, at a scan speed of 666-1428amu/s and event time 0.5 s. The identification of the chromatographic peaks was performed by comparing their mass spectra with those in the National Institute of Standards and Technology (NIST 05) library.

Molecular Docking Study

Protein-ligand interactions were studied with the help of PyRx by means of molecular docking simulations. PyRx is an online screening system that uses a combination of AutoDock Vina and AutoDock 4.2 (Lawal et al., 2025). The PubChem database (www.pubchem.com) was used to retrieve phytochemical compounds and three FDA-approved reference drugs in SDF format. These were converted to the pdbqt format for docking studies (Lawal et al., 2020; Gulati et al., 2023). Three bacterial enzymes, enoyl-acyl carrier protein reductase (ECPR), UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) and penicillin-binding protein (PBP) were selected as targets because they are essential metabolically and biosynthetically. Each enzyme had its own standard ligand for validation: Triclosan (PDB ID: 1D8A) for ECPR, Fospomycin (PDB ID: 3RK6) for MurA, and Amoxicillin (PDB ID: 3ITA) for PBP. These standards have been chosen as references to assess the affinities of the phytochemicals in the binding part, and they indicate their experimentally confirmed inhibitory potential.

Three-dimensional structures of the target proteins were downloaded from the Protein Data Bank (http://www.rcsb.org/pdb). The grid box analysis was used to identify active sites, focusing on the essential residues involved in ligand binding (Verma et al., 2023). Protein structures were optimized by eliminating unnecessary water molecules. Geometric optimization was performed to obtain appropriate docking conformations. Simulations of docking were performed in PyRx, and ligands were docked into predicted active sites, and binding affinities were obtained. The resulting protein-ligand interactions (hydrogen bonds, hydrophobic contacts, and other force stabilizers) were examined with the help of PyMOL and the Protein-Ligand Interaction Profiler (PLIP) to gain a better understanding of the binding modes and the effectiveness of the inhibitors (Spackman et al., 2021; Verma et al., 2022; Kumar et al., 2024).

ADME Properties and Toxicity Prediction

The physicochemical properties, lipophilicity, water solubility, pharmacokinetics, drug-likeness and toxicity prediction, including the violation of Lipinski rule of five (Lipinski et al., 1997) was studied using SwissADME online (http://www.swissadme.ch) (Lawal et al., 2020) and ProTox-3.0 online software (https://tox.charite.de/protox3/index.php?site) (Banerjee et al., 2024).

Data Analysis

The results of the conducted experiments were tabulated and summarised using descriptive statistics. The antibacterial activity was determined by measuring the inhibition zone diameter (mm) in three replicates and calculating the mean. Their direct comparison with ciprofloxacin results was used to determine the antibacterial activity of the extracts. To perform molecular characterization, sequence data from fungal isolates were compared using BLAST on the NCBI database to identify the species. The mass spectra of the compounds in the extracts were compared with those reported in the NIST library to identify them. The molecular docking results were evaluated based on binding affinity scores (kcal/mol) and interaction profiles. SwissADME and ProTox-3.0 predictions were used to determine pharmacokinetic and toxicological characteristics of the identified compounds. Tables, figures and descriptive summaries were used to state all interpreted data to facilitate comparative and inferential discussions.

RESULTS

Table 1: Identification of Endophytic Fungal Species Isolated from Moringa oleifera

Figure 1a: Macroscopic Features of Aspergillus niger Strain MOR-1

Figure 1b: Microscopic Features of Aspergillus niger Strain MOR-1

A description of both macroscopic and microscopic features of the isolated endophytic fungus was provided before molecular confirmation. The macroscopic features observed were downy white to yellow umbonate colonies with dark brown to black pinpoints and a white border, with a milk to pale-yellow reverse side (Figure 1a). Microscopically, the fungus exhibited branched, septate, hyaline hyphae with long, smooth conidiophores bearing large, globose vesicles surrounded by radiate, biseriate phialides (Figure 1b). These observed features were compared with those of already-identified fungal species to assess similarity. The macroscopic and microscopic features of the identified fungal species are also presented in Table 1.

Table 2: Molecular Identification of Endophytic Fungi Using BLASTn Search

Figure 2: Neighbour-Joining tree showing the evolutionary relationship between Aspergillus niger MOR-1 from this study and other similar isolates from NCBI GenBank

The molecular identification of the fungal organism was performed by blasting the sequenced nucleotides of the ITS1 and ITS4 regions against the NCBI database. The query sequence corresponded to the Aspergillus niger strain LA01, namely the 5.8S ribosomal RNA gene and the internal transcribed spacer 2 (ITS2), complete sequence; and the 28S ribosomal RNA gene, partial sequence (GenBank accession number: HQ392475.1). The percentage of similarity was 100. The phylogenetic tree generated using the maximum likelihood technique against ITS sequences of already known fungal species is illustrated in Table 2 and Figure 2, and the outcome affirmed the fungal organism to be related to the Aspergillus niger strain HQ392475.1

Table 3: Antibacterial Activity of Crude Extract of Aspergillus niger MOR-1

Antibacterial activity of the crude extract of the identified endophytic fungal isolate was measured as the mean zone of inhibition (mm) against the pathogenic bacterial strains (Table 3). Aspergillus niger MOR-1 appears to be widespread and a very effective antibacterial candidate, with the ability to produce an inhibition zone similar to that of ciprofloxacin against various pathogens. It inhibited S. aureus and S. pneumoniae with 21.67 ± 1.53 mm and 20.33 ± 1.53 mm zones of inhibition, respectively; slightly higher than the 20.6 ± 0.29 mm and notably lower than the 28.8 ± 0.38 mm zones recorded by the control for the respective bacteria. Yet it showed no effect on MRSA, which, interestingly, aligns with the inactivity observed in the control. More strikingly, it exhibited activity against P. aeruginosa (16.67 ± 1.53 mm) higher than the 12.10 ± 1.05 mm inhibition by the control, and activity on three Enterobacteriaceae: K. pneumoniae (29.33 ± 1.53 mm), S. typhii (29.33 ± 2.52 mm), and E. coli (28.33 ± 1.53 mm) all substantially higher than the control’s 24.6 ± 0.23 mm, 27.00 ± 1.00 mm, and 25.5 ± 0.47 mm respectively. The lack of inhibition of Shigella spp., Citrobacter spp., and Bacillus sp. hints at target-specific resistance traits, whereas the control exhibited substantial zones of 28.8 ± 0.38 mm, no activity, and 30.1 ± 0.41 mm, respectively, against these isolates.

Table 4: GCMS Result of Crude Extract of Aspergillus niger strain MOR-1

Pk Number	Area (%)	Compound CID	IUPAC Name	Molecular Weight (g/mol)	Molecular Formula	Chemical Structure
1	11.87	14259	Methyl eicosanoate	326	C21H42O2
2	46.66	21205	Methyl 14-methylpentadecanoate	270	C17H34O2
3	25.17	5354133	Methyl ricinoleate	312	C19H36O3
4	4.56	5362793	Methyl (13E,16E)-13,16-octadecadienoate	294	C19H34O2
5	1.44	5362805	Methyl (6E,9E,12E)-6,9,12-octadecatrienoate	292	C19H32O2
6	1.77	6421258	Arachidonic acid methyl ester		C21H34O2
7	0.89	5364432	Methyl (11E)-11-octadecenoate	296	C19H36O2
8	0.53	5365682	9,12-Octadecadien-1-ol, (Z,Z)-	266	C18H34O
9	7.11	8201	Methyl n-octadecanoate	298	C19H38O2

Figure 3: GC-MS Chromatogram of Aspergillus niger strain MOR-1

Analysis of crude extract of Aspergillus niger MOR-1 using Gas Chromatography-Mass Spectrometry (GC-MS) demonstrated various fatty acid methyl esters (FAMEs) and lipid derivatives to be the primary components of the fungi metabolic profile (Table 4 & Fig. 3). The chromatogram exhibited a number of peaks that represented structurally different aliphatic substances with molecular weights of between 266 and 326 g/mol. The range shows that long-chain fatty acid esters and unsaturated lipid molecules are preponderant. The data indicate that methyl eicosanoate (11.87%), methyl 14-methylpentadecanoate (46.66%), methyl ricinoleate (25.17%), methyl (13E,16E)-13,16-octadecadienoate (4.56%), methyl (6E,9E,12E)-6,9,12-octadecatrienoate (1.44%), arachidonic acid methyl.

Table 5: FTIR Result of Crude Extract of Aspergillus niger MOR-1

Figure 4: FTIR – Chromatogram of Aspergillus niger strain MOR-1

The FTIR spectrum of the crude extract of Aspergillus niger strain MOR-1 (Table 5 & Fig. 4) showed that it had a wide range of functional groups that were characteristic of its complicated biochemical structure. =C-H bending at 867.03 cm^-1 and =C-H stretching at 3074.63 cm^-1 was used to identify alkenes, and C-O and C-N stretching at 1071.49 cm^-1 and 1230.63 cm^-1, respectively, were used to identify ethers and amines. The CH2 bending at 1406.15 cm^-1 and CH2 stretching at 2885.60 cm^-1 indicated lipid or fatty acids, and the C-H stretching at 2820.02 cm^-1 indicated aldehydes. Nitro compounds were identified by N-O at 1523.82 cm^-1, proteinaceous or nitrogenous metabolites by amide C=O at 1633.76 cm^-1, and ketones by C=O at 1761.07 cm^-1. The O=C=O band detected the presence of carbon dioxide at 2342.62 cm^-1, and, at the same time, the presence of alcohols and hydroxyl groups was detected by the O-H band at 2633.89 cm^-1 and by an extensive range of other peaks at 3298.38-3855.83 cm^-1. Moreover, N-H amines were detected at 3140.22, 3218.34, and 3934.92 cm^-1, which, together, confirmed the presence of diverse biochemical components in the fungal extract.

Table 6: Molecular Docking Result of Bioactive Compounds from Crude Extract of Aspergillus niger (MOR-1) Against Bacterial Target Proteins

Molecular docking of the identified bioactive compounds in the crude extract of Aspergillus niger MOR-1 was performed to analyse their potential inhibitory effects on the bacterial essential enzymes ECPR, MurA, and PBPs. Table 6 shows that the docking scores of the studied compounds ranged from -4.0 to -6.2 kcal/mol, indicating that the fungal metabolites have favourable binding affinities for the active sites of the target proteins. The fungal compounds had greater or similar affinities when compared to the docking scores of the standards, ECPR (Triclosan (-5.2 kcal/mol)), MurA (Fosfomycin (-4.7 kcal/mol)) and PBPs (Amoxicillin (-4.8 kcal/mol)), indicating that the compounds hold promise in inhibitory ability. The most promising compound screened was methyl ricinoleate (CID 5354133), with the highest binding affinities with all targets (-6.0, -5.7, and -6.2 kcal/mol, respectively). Other compounds, including methyl (6E,9E,12E)-6,9,12-octadecatrienoate (CID 5362805) and methyl (11E)-11-octadecenoate (CID 5364432), were also found to have significant binding affinity, especially to PBPs and ECPR. The general docking trend shows that the fungal metabolites, which are mainly long-chain fatty acid esters and unsaturated lipids, have high affinity for bacterial enzymatic targets, increasing their promise as a potential source of natural antibacterial activity (Figure 5).

Table 7: Amino Acid Interaction Profiles of Selected Bioactive Compounds from Crude Extract of Aspergillus niger strain MOR-1 with Bacterial Target Proteins

Interaction analysis of selected bioactive compounds in the crude extract of Aspergillus niger MOR-1 with three major bacterial target enzymes, as illustrated in Table 7, reveals that the compounds exhibit strong binding affinities for the active-site residues of the target proteins. In the ECPR, the primary interactions involve residues ASN30A, TRP52A, ILE247A, and ILE251A. The ligands of MurA interacted with ARG91A, ALA115A, ARG120A, and ASP369A. It also forms hydrogen bonds with GLY114A and LEU370A, demonstrating stable, specific binding in the MurA active pocket. In the case of PBPs, several hydrogen bonds and hydrophobic interactions with key residues, including TYR580A, GLN582A, GLU545A, and LYS583A, are observed in their interaction profiles.

Figure 5: 3D docking pose interactions of ECPR, MurA and PBPs target proteins with bioactive compounds: (A) CID14259, (B) CID5354133, and (C) CID5362805 derived from extracts of Aspergillus niger strain MOR-1

Table 8: Drug-likeness, Lipophilicity, and Physicochemical Properties of Selected Bioactive Compounds from Crude Extract of Aspergillus niger strain MOR-1

[a] MW: molecular weight (<500, expressed as Dalton); [b] TPSA: Topological polar surface area (≤ 140 Ų ); [c] n-HBD: number of hydrogen bond donors (≤5); [d] n-HBA: number of hydrogen bond acceptors (≤10); [e] n-ROTB: number of rotatable bonds (≤ 10); [f] M Ref: molar refractivity (40–230); [f] LogP: logarithm of partition coefficient (<5) of compound between n-octanol and water; [g] LV: Lipinski’s violation; [i] Pre. LD50: Predicted LD50 (> 2000) (Nhlapho et al., 2024).

The data in Table 8 show the estimated drug-likeness, lipophilicity, and physicochemical key parameters of the active compounds identified in crude extracts of the Aspergillus niger strain MOR-1. Key parameters include molecular weight, topological polar surface area (TPSA), hydrogen bond donors and acceptors, rotatable bonds, molar refractivity, Log P, Lipinski violations, and predicted median lethal dose (LD₅₀). The compounds displayed molecular weights of 292.46–326.56 g/mol and TPSA values of 26.30–46.53 Ų. Hydrogen bond donors ranged from 0–1, acceptors from 2–3, and rotatable bonds from 14–19. Log P values ranged from 5.17 to 7.20, and each compound exhibited one Lipinski violation. Predicted LD₅₀ values ranged from 48 to 20,000 mg/kg. Reference threshold values for standard drug-likeness parameters are provided for comparison

Table 9: Toxicity Prediction of Selected Bioactive Compounds from Crude Extract of Aspergillus niger strain MOR-1

The data in Table 9 present the in-silico toxicity profiles of selected bioactive compounds from Aspergillus niger (MOR-1). The parameters include acute oral toxicity class, blood–brain barrier (BBB) permeability, gastrointestinal absorption, nutritional toxicity, carcinogenicity, hepatotoxicity, nephrotoxicity, neurotoxicity, human oral bioavailability, P-glycoprotein inhibition, and cytochrome P450 (CYP) isoform inhibition.

The predicted acute oral toxicity of the identified compounds ranged between Class V and Class VI, indicating low acute toxicity. All compounds demonstrated positive blood–brain barrier permeability and high gastrointestinal absorption, while none showed predicted nutritional toxicity, carcinogenicity, hepatotoxicity, nephrotoxicity, or neurotoxicity. Human oral bioavailability was predicted to be negative for all compounds. With respect to metabolic interactions, two compounds (CIDs 14259 and 5354133) were predicted to inhibit P-glycoprotein, whereas CID 5362805 showed no P-glycoprotein inhibitory activity. Limited cytochrome P450 inhibition was observed, with CID 5362805 predicted to inhibit CYP1A2 and CYP2C19, while no inhibitory effects were predicted for CYP2C9, CYP2D6, CYP3A4, or CYP2E1 across all compounds.

DISCUSSION

Moringa oleifera offers a chemically diverse platform on which endophytic fungi with potential antibacterial activity can be selected and characterized. Particular attention is given to leaves for both practical and scientific reasons, such as their primary role in secondary metabolism and their value as hosts for microbial symbionts (Pang et al., 2021). Recent phytochemical and microbiological data are supported by the plant's ethnopharmacological history and thus confirm the hypothesis that its endophytes could produce new bioactive substances. Therefore, the host plant is scientifically justified as a potential source for bioprospecting and the identification of natural products, consistent with its traditional use as medicine.

Following isolation, antibacterial screening against ten clinical bacterial isolates revealed detectable inhibitory activity, highlighting the selective nature of antibacterial metabolite production by the endophytic fungi. Zones of inhibition were quantified as mean ± SD, and comparative analysis against a standard antibiotic control provided a framework for assessing relative potency, spectrum, and limitations. The observed variability in antibacterial activity reflects differences in fungal secondary metabolite composition and in bacterial structural defences, including outer membrane permeability and intrinsic resistance mechanisms.

Within this context, Aspergillus niger strain (MOR-1) exhibited a broad and potent antibacterial spectrum. Inhibition against S. aureus and S. pneumoniae was comparable to the control. Notably, MOR-1 demonstrated activity against P. aeruginosa, K. pneumoniae, S. typhi, and E. coli, exceeding control values in each case. The lack of activity against MRSA and certain enteric species suggests the presence of strain-specific resistance mechanisms. The activity against S. pneumoniae aligns closely with the findings of Baz et al. (2024), who reported an inhibition zone of 18 ± 0.11 mm for A. niger culture filtrate extract. Notably, the activity against S. aureus observed in this study is more than double the 10.5 ± 0.08 mm inhibition reported by Baz et al. (2024), underscoring the extract's greater potency. The inhibition zone on E. coli exceeds the 23.5 ± 0.06 mm observed by Baz et al. (2024), indicating a stronger antibacterial effect.

The inhibition of S. aureus concurs with the 15.66 ± 0.57 mm (Rani et al., 2017), 18.39 ± 0.03 mm (Niazi et al., 2023), and 10.157 ± 0.79 mm (Satriawan et al., 2025) reports, but contradicts the total inactivity noted by Fuego et al. (2021). Similarly, the absence of MRSA activity conflicts with Satriawan et al. (2025) (12.57 ± 1.09 mm). The P. aeruginosa zone aligns with Enany et al. (2022) (16.5 ± 0.71 mm) and Ramesh et al. (2022) (19.33 mm), and is virtually identical to the 17.0 ± 0.18 mm of Baz et al. (2024), yet surpasses the 3.8 ± 0.8 mm reported by Ababutain et al. (2021). The activity against K. pneumoniae and S. typhii is consistent with earlier reports (Rani et al., 2017; Khattak et al., 2021; Ramesh et al., 2022) but markedly exceeds the diameters reported by Ababutain et al. (2021) and Niazi et al. (2023). Finally, the lack of activity against Bacillus spp. contrasts with the 22.35 ± 0.20 mm inhibition of B. subtilis found by Niazi et al. (2023) and the activity recorded by Fuego et al. (2021). The exceptional potency of MOR-1 likely arises from a synergistic mixture of low-molecular-weight polyketides and ionophore-like compounds capable of disrupting proton gradients and membrane integrity (Rani et al., 2021; Liu et al., 2025).

In support of this observation, FTIR and GC–MS results indicated a lipid-centric profile, suggesting the abundance of hydroxylated and unsaturated fatty acid esters. These amphiphilic molecules can integrate into bacterial membranes, induce oxidative stress, and impair enzymatic function (Harayama and Antonny, 2023; Trabelsi et al., 2023; Jomova et al., 2023). The convergence of spectroscopic and chromatographic data supports the extract’s pronounced antibacterial efficacy and ecological fitness.

At the molecular level, docking analysis revealed favourable binding of selected compounds to ECPR, MurA, and PBPs, enzymes essential for bacterial lipid synthesis and cell wall integrity. Compounds from A. niger strain MOR-1 consistently exhibited strong affinities (up to −6.2 kcal/mol), likely due to optimal hydrophobic complementarity within enzyme pockets (Abdellatief et al., 2025; Alanazi, 2025). Protein–ligand interactions were stabilized by hydrogen bonding and hydrophobic contacts involving catalytically relevant residues, thereby validating the docking predictions.

Furthermore, ADME/T profiling indicated that all selected compounds comply with Lipinski’s rule of five and exhibit favorable pharmacokinetic properties. Most compounds belonged to low-toxicity classes (V–VI), with minimal CYP450 and P-gp inhibition, suggesting a low risk of drug–drug interactions and efflux-mediated resistance (Cheng et al., 2012). Collectively, these attributes support their candidacy for further preclinical development.

CONCLUSION

Moringa oleifera leaves provide a scientifically validated platform for screening for antibacterial endophytic fungi. The endophytic strain Aspergillus niger MOR-1 demonstrated broad and significant antibacterial activity against various clinically relevant pathogens, often equaling or surpassing standard antibiotic treatments, though with specific limitations across strains. Analyses using spectroscopy and chromatography indicated a metabolite profile high in lipids, primarily consisting of amphiphilic fatty acid derivatives, which support membrane-disruptive antibacterial mechanisms. Molecular docking studies revealed strong, stable interactions between selected compounds and key bacterial enzymes, while ADME/T predictions indicated favourable drug-like properties and minimal toxicity. Altogether, these observations position A. niger strain MOR-1 as a potential source of new antibacterial agents and encourage further purification and preclinical investigation.

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