Bioassay-guided fractionation and biological activities of antimycin A and 4-hydroxybenzoic acid isolated from Nocardiopsis sp. strain LC-9

Abstract

Nocardiopsis sp. strain LC-9 was isolated from freshwater sediments and explored for its varied bioactive traits. Initially, ethyl acetate extract of strain LC-9 at varied concentrations showed pronounced antibacterial activities. After column chromatography, fraction F2 and F3 of the extract were identified as prominent fractions in terms of antimicrobial activities with low minimum inhibitory concentration values. Antioxidant activities of fraction F2 and F3 revealed remarkable scavenging of free radicals with low IC₅₀ values (DPPH – 417.86 ± 0.24 μg/mL, ABTS – 431.6 ± 0.90 μg/mL, and FRAP – 404.36 ± 0.18 μg/mL). Fractions F2 and F3 were further characterized by UV spectrum, Fourier transform infrared spectroscopy, nuclear magnetic resonance, and liquid chromatography-mass spectrometry, and were identified as Antimycin A and 4-hydroxybenzoic acid. The compounds were further tested for anticancer activity against MCF-7 cells. The MTT assay showed reduced viability of MCF-7 cells with an increase in concentration of compounds. The IC₅₀ values for Antimycin A and 4-hydroxybenzoic acid were 9.6 ± 0.7 μg/mL and 20.8 ± 0.4 μg/mL, respectively. Staining techniques confirmed the apoptosis mechanism. Finally, molecular docking (against targeted proteins of bacteria, fungus, and cancer cells) and molecular dynamics confirmed the pharmaceutical efficacy of the purified compounds.

Full Article

Bioassay-guided Fractionation and Biological Activities of Antimycin A and 4-Hydroxybenzoic Acid Isolated from Nocardiopsis sp. Strain LC-9

Rozario Sagaya Jansi,^a Ameer Khusro,^b,* Paul Agastian,^c,* Mikhlid H. Almutairi,^d and Bader O. Almutairi ^d

Nocardiopsis sp. strain LC-9 was isolated from freshwater sediments and explored for its varied bioactive traits. Initially, ethyl acetate extract of strain LC-9 at varied concentrations showed pronounced antibacterial activities. After column chromatography, fraction F2 and F3 of the extract were identified as prominent fractions in terms of antimicrobial activities with low minimum inhibitory concentration values. Antioxidant activities of fraction F2 and F3 revealed remarkable scavenging of free radicals with low IC₅₀ values (DPPH – 417.86 ± 0.24 μg/mL, ABTS – 431.6 ± 0.90 μg/mL, and FRAP – 404.36 ± 0.18 μg/mL). Fractions F2 and F3 were further characterized by UV spectrum, Fourier transform infrared spectroscopy, nuclear magnetic resonance, and liquid chromatography-mass spectrometry, and were identified as Antimycin A and 4-hydroxybenzoic acid. The compounds were further tested for anticancer activity against MCF-7 cells. The MTT assay showed reduced viability of MCF-7 cells with an increase in concentration of compounds. The IC₅₀ values for Antimycin A and 4-hydroxybenzoic acid were 9.6 ± 0.7 μg/mL and 20.8 ± 0.4 μg/mL, respectively. Staining techniques confirmed the apoptosis mechanism. Finally, molecular docking (against targeted proteins of bacteria, fungus, and cancer cells) and molecular dynamics confirmed the pharmaceutical efficacy of the purified compounds.

DOI: 10.15376/biores.19.4.7673-7697

Keywords: Nocardiopsis sp.; Antimycin A; 4-Hydroxybenzoic acid; MCF-7; Molecular docking; Molecular dynamics

Contact information: a: Department of Bioinformatics, Stella Maris College, Chennai-600086, Tamil Nadu, India; b: Department of Research Analytics, Saveetha Dental College and Hospitals, Saveetha Institute of Medical and Technical Sciences, Saveetha University, Chennai – 600077, India; c: Research Department of Plant Biology and Biotechnology, Loyola College, Chennai-600034, Tamil Nadu, India; d: Department of Zoology, College of Science, King Saud University, P.O. Box 2455, Riyadh 11451, Riyadh, Saudi Arabia;

*Corresponding author: armankhan0301@gmail.com; agastianloyolacollege@gmail.com

INTRODUCTION

Research in discovering actinomycetes and screening their biological activity is a major field of interest in India. Over the past three decades, actinomycetes have substantiated themselves as dependable sources of therapeutically useful bioactive metabolites (Saraswathi et al. 2020). The compounds display a comprehensive scale of bioactivities, such as antimicrobial, anticancer, cytotoxic, neurotoxic, antiviral, and antineoplastic activities (Selim et al. 2021).

It has been reported that of the entire microbial metabolites in the globe, 70% is contributed by actinomycetes, while 20% by fungi, 7% by Bacillus sp., and 1 to 2% by Pseudomonas sp. This substantiates that actinomycetes stand out exclusively in the production of an array of biologically active metabolites for clinical research (Hauhan and Gohel 2020).

Of the antibiotic-producing microorganisms, actinomycetes still remain the major constituent of diverse bioactive and pharmaceutically distinct metabolites. Different classes of antibiotics with varying target mechanisms and unique mode of action, such as amino-glycosides, polyketides, chloramphenicols, tetracyclines, etc., are produced by the class Actinobacteria. Molecular data, such as 16S rRNA sequencing, have been beneficial in actinomycetes classification and identification (Salwana and Sharma 2020).

Soil inhabiting Nocardiopsis sp. belongs to the phylum Actinobacteria, containing a large repository of unique bioactive compounds. The genus Nocardiopsis (Nocardiopsaceae) was first defined by J. Meyer in 1976. Morphological characters, phylogeny, and chemotaxonomic properties of the species articulate a discrete ancestry from the order ‘Actinomycetales’. This genus comprises of Gram positive aerobic actinomycetes with aerial mycelium that fragments into spores and form wrinkled/folded colonies on organic media (Abdel-Razek et al. 2020; Dror et al. 2020)

Nocardiopsis is reported to contain 24 species and two subspecies, mostly thriving in alkaliphilic or halophilic environments. The prominent features of the cell wall of this genus include: meso-2,6-diaminopimelic acid (cell-wall chemotype III), phosphatidyl-choline as a characteristic phospholipid (phospholipid type III), menaquinones, absence of madurose and nocardomycolic acids, and novel teichoic acid compositions. The members of this species generally have genomes with a high load of G+C (64 to 71%) composition (Ngema et al. 2023). Nocardiopsis spp. are reported in diverse regions including saline, hypersaline, desert, and alkaline environments (Ramalingam et al. 2022; Kumar et al. 2023).

Microbial metabolites account for 45% of the world market of pharmaceuticals. The marketing of the different classes of products reaches millions of dollars each year. In recent times, discovery of novel compounds with bioactive potential has become rare and remains a great challenge. This provokes recent researchers to find new species (genera or families) with altered and novel genes to produce distinct metabolites of new purpose. In this investigation, a significant attempt was undertaken to assess the biological activities of bioactive metabolites isolated from Nocardiopsis sp. strain LC-9.

EXPERIMENTAL

Sample Site, Sampling Procedure, and Isolation

Freshwater soil sediments were collected from Chembarambakkam lake at a latitude of 13° 00′ 41.69″ N and longitude of 80° 03′ 38.27″ E Chennai, Tamil Nadu, India. Actinomycetes were isolated from the collected soil samples using serial dilution technique on Actinomycetes Isolation Agar (AIA) medium augmented with actidione (20 mg/L) and nalidixic acid (100 mg/L). Plates were incubated at 28 ± 2 °C for 7 to 10 days. After the required incubation period, pure culture of isolates was obtained using the streaking method and stored at 4 °C (Fig. 1).

Fig. 1. a) Freshwater soil sediments collection site (Chembarambakkam lake), b) Actinomycetes colonies isolated on AIA medium using serial dilution method, and c) Pure culture of isolate

Preliminary Screening by Perpendicular Streak Method

Primary screening was performed by the cross-streak method on Mueller Hinton Agar media against 11 bacterial pathogens. This was done by inoculating a single streak of the pure culture of isolates in the center of the Petri plates and stored at 28 ± 2 °C for 5 days. Successively, the test pathogens were streaked at 90° angle and incubated at 37 °C overnight. Lack of growth of pathogens indicates antibacterial activity of isolates.

Morphological and Molecular Characterization of Potent Isolate

Morphological and cultural features of the potent isolate was observed by inoculating and incubating the culture in various media including AIA, MNGA, SCA, NA, M5, ISP1, ISP2, ISP3, and ISP4 aseptically at 28 °C for 7 to 14 days. The isolate was subjected to smear preparation for Gram staining. The spore chain characteristic of the selected isolate was observed by scanning electron microscopy (SEM; JSM5600LV, JEOL, Japan). For efficient genomic DNA isolation, Hipura Streptomyces DNA Purification Kit (Hi-media, India) was used. DNA was eluted and visualized by 1% agarose gel electrophoresis. Polymerase chain reaction (PCR) amplification was performed using the following universal primers: 27F (5`AGTTTGATCCTGGCTCAG3`) and 1492R (5`ACGGCTACCTTGTTACGACTT3`). Sequencing was achieved using Applied Biosystems, (Foster City, CA) USA, and the standard quality of the sequence was checked using sequence Scanner Software v1 (Applied Biosystems, Foster City, CA, USA). Similarly, the sequence alignment and editing were carried out by Geneious Pro v5.6 (http://www.geneious.com). Finally, a phylogenetic tree was constructed using MEGA X software (https://www.megasoftware.net/) for the target sequence.

Antibacterial Activity of Crude Extract

The isolate was inoculated into modified nutrient glucose broth medium and allowed to ferment for 7 days at 28 °C at 180 rpm. After fermentation, the culture filtrates were extracted by liquid-liquid extraction method using ethyl acetate. Antibacterial activity of the extract at varied concentrations (1.25 to 5 mg/mL) was tested using the disc diffusion method. Streptomycin was used as a positive control.

Bioactive Metabolites Extraction by Column Chromatography

The crude ethyl acetate extract was separated by silica gel column (4.5 to 60 cm) chromatography. A total of 10 g of the extract were used for the column preparation along with silica gel (65 to 70 g). The column initially used ethyl acetate and was gradually added with increasing the polarity mixtures of solvents of hexane and ethyl acetate (0 to 100%). Four fractions (F1, F2, F3, and F4) were collected at regular intervals and spotted in TLC plates with the same solvent proportion to find the Retention factor (Rf) values. Concurrently, fractions with the same Rf values were combined and evaporated to remove the solvents at appropriate conditions.

Minimum Inhibitory Concentration (MIC) Determination

The collected fractions were tested for antimicrobial activity against 11 bacterial pathogens [Bacillus subtilis (MTCC 441), Staphylococcus aureus (ATCC 25923), MRSA Clinical pathogen (15DR), S. aureus (Methicillin sensitive), Enterobacter faecalis (MTCC 29212), Escherichia coli (MTCC 40), Klebsiella pneumoniae (MTCC 109), Enterobacter aerogenes (MTCC 111), Vibrio parahaemolyticus (MTCC 451), Yersinia enterocolitica (MTCC 840), and Pseudomonas aeruginosa (ATCC 15380)] and 5 fungal cultures [Aspergillus flavus (MTCC 9390), Aspergillus niger (MTCC 478), Botrytis cinerea (MTCC 359), Candida albicans (MTCC 227), and Candida lunata (MTCC 2098)] via the microdilution method (Venkatadri et al. 2017). All the pathogens were obtained from Entomology Research Institute, Loyola College, Chennai, India. Standard streptomycin and fluconazole were used as bacterial and fungal positive control, respectively.

Antioxidant Properties of Active Fractions

The DPPH (2, 2, diphenyl-1-picryl hydrazyl) radical scavenging, ABTS [2, 2′-azinobis (3-ethylbenzothiazoline-6-sulfonic acid)] radical scavenging, FRAP (ferric reducing antioxidant power) activity, and total reducing power assay of the most active fractions (F2 and F3) were determined as per the methodology of Oyaizu (1986), Benzie and Strain (1996), Jia et al. (1999), Kekuda et al. (2010), and Prasathkumar et al. (2021). The IC₅₀ values were calculated by online linear regression curve.

Structure Elucidation of Active Fractions

Based on the antimicrobial and antioxidant results of fractions, fraction F2 and F3 were selected and further characterized by UV spectrum (JASCO, model V-750, Japan), Fourier transform infrared (FT-IR) spectrum (Bruker, Alpha-T ATR-FTIR Spectrometer model, TENSOR 27, Germany) with the spectra characteristics detected at 20000 to 500 cm^-1 range, ¹H and ¹³C nuclear magnetic resonance (NMR) (Bruker, model AVANCE III 500 NMR, Germany), and liquid chromatography-mass spectrometry (LC-MS).

Cytotoxicity Assay of Isolated Compounds

In vitro cytotoxic activity of the compounds was tested against human breast cancer cell line (MCF-7) procured from the National Center for Cell Sciences (NCCS), Pune, India. The cells were incubated at 37 °C with 5% CO₂ in a humidified CO₂ chamber. The cytotoxicity was determined using MTT [3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide] assay as per the methodology of Mosmann (1983). The percentage of viability was calculated as:

% viability = [Absorbance of sample/Absorbance of control] × 100

Apoptotic Cell Death Analysis

The apoptotic cells were observed by staining the cells with 1 µL of 100 mg/mL of acridine orange (AO) and 100 mg/mL of ethidium bromide (EtBr) in distilled water. To this, 90 µL of cell suspension (1 × 10⁵ cells/mL) were added. Finally, after staining with 10 μL of DAPI (4′, 6-diamidino-2-phenylindole), the samples were observed under fluorescent microscope (Nikon Eclipse, Inc. Tokyo, Japan) (Clarance et al. 2020).

Molecular Docking Analysis and ADME Prediction

N-myristoyl transferase (1IYL) and 14-α-demethylase (5FSA) were used as targeted proteins for fungi. Dihydropteroate synthetase (2VEG), Isoleucyl- tRNA Synthetase (1ILE), Alanine racemase (2RJG), Penicillin-binding protein 1a (3UDI), and Dihydrofolate Reductase (3SRW) were used as targeted proteins for bacteria. In contrast, vascular endothelial growth factor receptor 2 (5EW3), B-cell lymphoma 2 (5VAX), DNA topoisomerases II (1ZXM), HER2-human epidermal growth factor receptor 2 (5O4G), and EGFR (Epidermal Growth Factor Receptor; 2J6M) were used as targeted proteins of cancer cell line. Docking was performed between the compound and selected proteins using Schrödinger software (version 2020-1; Maestro, Schrödinger, LLC, New York, NY, USA). The ADME (absorption, distribution, metabolism, and excretion) likelihood was estimated using the Qik prop tool to evaluate the lead likeliness and Lipinski violations.

Molecular Dynamics (MD) Simulations

Molecular dynamics simulations were run by Desmond v3.6 Package (Schrödinger, New York, NY, USA) to determine the stable conformation of the protein-ligand complex and effectiveness of the compound against the target protein. The studies were performed for 100 ns simulation to determine the interactions and calculate the Root Mean Square Deviation (RMSD), Root Mean Square Fluctuation (RMSF), and contact points.

Statistical Analysis

All the experiments were performed in triplicate and results were recorded as Mean ± SD. ANOVA was used to analyze the statistical significance and values with P≤0.05 were considered significant.

Fig. 2. Screening for antibacterial activity of isolate LC-9. The isolate showed inhibition of pathogen growth. 1 – B. subtilis (MTCC 441), 2 – S. aureus (ATCC 25923), 3 – MRSA Clinical pathogen (15DR), 4 – S. aureus (Methicillin sensitive), 5 – E. faecalis (MTCC 29212), 6 – E. coli (MTCC 40), 7 – K. pneumoniae (MTCC 109), 8 – E. aerogenes (MTCC 111), 9 – V. parahaemolyticus (MTCC 451), 10 – Y. enterocolitica (MTCC 840), 11 – P. aeruginosa (ATCC 15380).

RESULTS AND DISCUSSION

Screening and Identification of Nocardiopsis sp.

Figure 2 illustrates primary screening for antibacterial activity of isolate LC-9. Results showed that the growth of all the tested bacterial pathogens (horizontally streaked towards the circumference of the petriplate) was inhibited in the presence of isolate LC-9 (longitudinally streaked at the center of the petriplate), indicating prominent antibacterial activity of isolate LC-9.

Further, the morphological characteristic of isolate LC-9 by Gram staining and SEM analysis showed aerial mycelium and hyphae of various lengths (Fig. 3).

Fig. 3. A) Gram staining and B) SEM analysis of isolate LC-9

Molecular characterization, followed by phylogenetic tree analysis indicated that the isolate belonged to the genus Nocardiopsis, and the strain was designated as Nocardiopsis sp. strain LC-9 (Accession No. – MH266471). The optimal tree with the branch length of 499.08910173 is shown in Fig. 4. The tree was inferred from bootstrap consensus analysis (100 replicates) with a total of 1535 positions.

The genus Nocardiopsis from Actinobacteria plays a significant role in the production of secondary metabolites, especially of pharmacological importance. They thrive in extreme environments with high metabolic activity, thereby withstanding and resisting attacks of other pathogens. Several studies showed that extracts of Nocardiopsis have the capability to produce natural products with unveiled cytotoxic (Siddharth et al. 2021) and antimicrobial competence (Goel et al. 2021).

Ethyl acetate extract of strain LC-9 presented potential antibacterial activity against the test pathogens. The extract at 5 mg/mL concentration revealed a maximum zone of inhibition of 18.5 ± 0.16, 18.5 ± 0.28, 18.16 ± 0.57, 17.5 ± 0.28, and 17.16 ± 0.88 mm against B. subtilis, Y. enterocolitica, V. parahaemolyticus, E. aerogenes, and S. aureus (Methicillin sensitive), respectively (Table 1).

Fig. 4. Phylogenetic tree of strain LC-9

Antibacterial Activity of Strain LC-9 Extract

Table 1. Antibacterial Activity of Ethyl Acetate Extract of Strain LC-9

Each value represents the mean ± SD of triplicate experiments. ^abcdValues are significantly different (P<0.05). ‘-‘: Indicates lack of antibacterial activity.

MIC Determination of Strain LC-9 fractions

Among 4 fractions tested, fraction F2 of strain LC-9 exhibited promising antimicrobial activities against the indicator pathogens with minimum MIC value of 15.625 μg/mL (against MRSA clinical pathogen and K. pneumoniae) and 31.25 μg/mL (against Curvularia lunata). Likewise, fraction F3 showed potential MIC value of 31.25 μg/mL against MRSA clinical pathogen, S. aureus (Methicillin sensitive), K. pneumoniae, V. parahaemolyticus, Y. enterocolitica, and Aspergillus niger (Table 2).

Antioxidant Activity of Strain LC-9 Fractions

Antioxidant activities of fractions F2 and F3 are shown in Fig. 5. With increasing concentration (200 to 1000 μg/mL), the fractions showed approximately 38 to 80% of scavenging activities compared to the standards. IC₅₀ values of fraction F2 were estimated as 417.86 ± 0.24, 431.6 ± 0.90, 404.36 ± 0.18, and 416.16 ± 1.54 μg/mL towards DPPH scavenging, ABTS scavenging, FRAP assay, and total reducing power assay, respectively. In contrast, IC₅₀ values of fraction F3 were estimated as 321.72 ± 0.18, 333.71 ± 1.54, 414.67 ± 1.36, and 401.67 ± 1.39 μg/mL towards DPPH scavenging, ABTS scavenging, FRAP assay, and total reducing power assay, respectively.

Table 2. MIC Values of Fractions (F1, F2, F3, and F4)

‘-’: No activity

Characterization of fraction F2 and F3

The UV spectrum of the fraction F2 (compound 1) showed peak at 219 to 226 nm (Fig. 6A). Similarly, the FT-IR spectrum at 3419 cm^-1 revealed the presence of O-H or N-H groups. The peak at 2927 to 2974 cm^-1 showed C-H stretching in -CH₃ or -CH₂. The peak at 1746 cm^-1 described the C=O stretching, peak at 1534 to 1540 cm^-1 indicated the presence of C-O-C of aromatic ring, and the peak at 1050 cm^-1 specified alkyl group (Fig. 6B).

The ¹H NMR spectra of compound 1 had the following spectrum: at 0.874 ppm is the (3H, t), 1.116 to 1.207 ppm (2H, m), 1.305 to 1.318 ppm (3H, d), 2.493 to 3.326 ppm (CH₃, 3H, s), 4.856 to 4.996 ppm (aromatic, 1H, m), 5.311 to 5.342 ppm (aromatic, 1H, d), 5.565 to 5.618 ppm (aromatic, 1H, d), 6.901 to 6.931 (NH, 1H, d), 7.870 to 7.854 (NH, 1H, d), 8.237 to 8.326 ppm (CHO, 1H, d), 12.765 ppm (OH, 1H, s), respectively. The ¹³C NMR spectra showed the presence of methyl (CH₃) group in the upfield region between 39.538 and 40.540 ppm. The splitting patterns of both the NMR studies designated good evidence that compound 1 was identified as Antimycin A (Fig. 7A and 7B). Through the mass spectrum, the molecular weight (549.28 g/mol) and molecular formula (C₂₈H₄₀N₂O₉) of the compound were deduced in the study. The structure of the compound was elucidated and predicted to be Antimycin A of the subtype Antimycin A_1ab (Fig. 8).

Fig. 5. a) DPPH, b) ABTS, c) FRAP, and d) Total reducing power activity of standard and fractions (F2 and F3) at varied concentrations. Each value presents the mean ± SD of triplicate experiments.

Antimycin A is an antibiotic made of nine-membered dilactone ring. In 1949, it was first isolated and categorized as a fungicide from Streptomyces sp. (Han et al. 2012). So far, more than 20 Antimycin A derivatives have been reported. Class of Antimycin compounds generally express antibacterial and antifungal properties due to the presence of biosynthetic gene clusters like non-ribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs) in actinomycete strains. Detection methods like PCR amplification (for PKSs and NRPSs genes), NMR, and LC-MS were used to identify and categorize the different types of antimycin (Hytti et al. 2019).

Antimycin A plays an important role in the electron transport chain by inhibiting complex III. It also increases cell death and is the reason for the downfall of oxidative phosphorylation (Kauppinen 2018). In this study, Antimycin A isolated from strain LC-9 showed good antimicrobial and high antioxidant potential. Previously, two novel antimycins, Urauchimycins A and B, were obtained from Streptomyces sp. strain Ni-80, which showed antifungal activity against C. albicans. Likewise, there was an effective antifungal activity of Antimycins A₁₉ and A₂₀ from S. antibioticus H74-18 against C. albicans. Wherein, Antimycin B2 from S. lusitanus XM52 showed antibacterial activity against S. aureus and L. hongkongensis (Imamura et al. 1993; Han et al. 2012).

Fig. 6. A) UV visible spectroscopy and B) FT-IR spectroscopy of Antimycin A