Claviceps purpurea, Antibacterial, ADMET, DNA Gyrase, Molecular docking.

Ergot is a fungal disease caused by fungus of the genus Claviceps.  Species in this genus are unique in that they only infect ovaries of the host plants, no other part of the plant is infected. There are approximately 40 species of Claviceps with C. purpurea (Fries) Tulasne being the species of greatest concern (Schumann and Uppala 2000).

Ergot alkaloids, named after the ergot fungus Claviceps purpurea, can infect grains and cause epidemics, especially during the Middle Ages (Gerhards et al., 2014).

Ergot Claviceps purpurea (Fries) Tulasne is of critical economic importance because it is a producer of many biologically active compounds (alkaloids) for the pharmaceutical industry, a unique model of the parasite-host system, and a mycotoxin-associated pathogen that causes significant economic damage to agriculture around the world (Volnin et al., 2024).

Ergot alkaloids show strong interactions with serotonin, dopamine and adrenergic receptors of the central nervous system and also with adrenergic receptors in blood vessels. Therefore, they can act as potent drugs. Examples with pharmaceutical applications are, methylergometrine used in gynecology to stop bleeding after childbirth, ergotamine used to treat vascular migraine headaches, Parkinson's disease (Paul and Schiff  2006). 

Several studies point to the various activities of phytochemicals, antioxidant, cardioprotective, hepatoprotective, which are: antimicrobial, anti-inflammatory, analgesic, anti-hemorrhagic, antitussive, antitumor, immunostimulating, anticancer, antiviral, among other. Among these, some studies attribute considerable antimicrobial activity to phytochemicals commonly found in plants and microorganisms (Dantas et al., 2015).

Fungi provide a plentiful and diverse source of unique and often bioactive metabolites, and they have produced a number of medicinally important compounds, including penicillin, mevinolin, fingolimod, and caspofungin (VanderMolen et al., 2013).

Over the past decade, much attention has been placed on the study of phytochemicals for their antibacterial activity, especially against multidrug-resistant Gram-negative and Gram-positive bacteria. Antibiotic resistance, a major global health concern, is a result of the emergence of multidrug-resistant bacteria. This has led to the need for new, effective antibacterial agents to combat the problem. However, the process of discovering new antibiotics is costly and time-consuming, taking approximately ten years to bring a new antibiotic to market (Borges et al., 2015).

The process of finding new antibiotic drugs heavily relies on the in silico prediction of ADMET characteristics. These days, molecules with poor absorption, distribution, metabolism, and elimination (ADME) characteristics are removed from the drug development pipeline early on in the process, which significantly reduces research and development expenditures. Many people employ Lipinski's "Rule of Five" as a filter for qualities similar to drugs (Lajiness et al., 2004). Molecular docking is a commonly used method for evaluating the complex formation of small ligands with large biomolecules (Rudnitskaya et al., 2010). Insight of the above, the present study was undertaken to isolate and characterize antibacterial compounds from the in vitro derived ethyl acetate extract of Claviceps purpurea fungus and to verify the antibacterial property against pathogenic bacterial isolates (Shiva et al., 2018).

Preparation of fungal Extract. Prepare potato dextrose broth, then autoclaved at 121 ℃ and 15lbs pressure. After sterilization, a loopful of fungal inoculum was inoculated into the broth and incubated in rotary shaker at 28ºC for 3-4 days. After incubation, using Whatmann filter paper 1 for separation of filtrate. Using separating funnel, the culture filtrate was exposed to solvent extraction with ethyl acetate. Three repetitions of the experiment were conducted again. In a desiccator, the compound was allowed to air dry.

Preliminary phytochemical screening. Using the conventional techniques outlined by (Harborne, 2005), a preliminary phytochemical study of the fungal extract of Claviceps purpurea was performed to see whether any desirable secondary metabolites were present.

HR-LCMS analysis of fungal extract. The bioactive components of ethyl acetate extract from Claviceps purpurea fungus were analyzed using a High-Resolution Liquid Chromatograph Mass Spectrometer (HR-LCMS) G6550A system. The method used was 30 mins ± ESI 10032014_MSMS.m, and the gas temperature was 250°C. The compounds were identified by comparing their retention time and mass with a stored metlin library from IIT, Bombay (Shivakumar et al., 2018).

Antibacterial activity - Agar well diffusion assay. The study tested the antibacterial activity of aqueous and solvent extracts using an agar well diffusion method. The bacterial culture was spread on nutrient agar plates, and the extract was dissolved in DMSO at different concentrations. Wells were made on the plates, and 20 μl of each concentration of fungal extract was introduced. A positive control was ciprofloxacin (20 μg/ml). The plates were incubated for 24 hours at 37°C. The antibacterial activity was evaluated by measuring the growth inhibition zone for the test organisms compared to the control. The activity index was calculated to compare the zone of inhibition with the standard antibiotic (Pradeepa et al., 2014).

Minimum Inhibitory Concentration (MIC). The study confirmed antibacterial activity by determining the minimum inhibitory concentration (MIC) using microdilution method with resazurin. Bacterial suspensions were prepared using the direct colony method, with initial suspensions containing 106 CFU/ml. A twofold serial dilution of ethyl acetate fungal extract was made in Mueller-Hinton broth, and a final concentration of 5 x 106 CFU/ml was added to each well. Resazurin solution was added to each well to display microbial growth. The inoculated plates were incubated at 37°C for 24 hours. The MIC was defined as the lowest concentration that prevented resazurin color change from blue to pink. ANOVA was performed using ezANOVA software and Microsoft excels to determine the mean and standard error (Nikolic et al., 2014).

Molecular docking studies. The Lipinski "Rule of five" is used to filter drug-like properties, and in silico pharmacokinetic properties and ADME (absorption, distribution, metabolism, and elimination) and toxicity analysis were predicted using Data Warrior. The chemical structure of identified compounds, Aldicarb, Arecoline, Glimepiride, Gedunin, and Pentobarbital, and the standard Drug ciprofloxacin, were drawn using Chem Bio Draw tool. The energy of each molecule was minimized using ChemBio3D, and the energy minimized ligand molecules were input for AutoDock Vina for docking simulations. The protein data bank coordinate file was used as the receptor molecule, and the docking algorithm was used to search for the best-docked conformation between ligand and protein (Reece and Maxwell 1991; Bax et al., 2010; Trott and Olson 2010; Laskowski and Swindells  2011).

Preparation of ethyl acetate extract. Claviceps purpurea filtrate was placed in separating funnel then add ethyl acetate solvent shake it for mixed thoroughly, then allowed for few minutes the secondary metabolites are settled in top layer, easily remove and collect the top layer and allowed for solidification (Fig. 1).

Fig. 1. Preparation of ethyl acetate extract of Claviceps purpurea.

Preliminary Phytochemical analysis. The preliminary phytochemical analysis of ethyl acetate extract of Claviceps purpurea showed a positive result for alkaloids, tannins, steroids, glycosides and terpenoids. The results are presented in Table 1 and Fig. 2.

Table 1: Preliminary phytochemical analysis of ethyl acetate extracts of C. purpurea.

Fig. 2. Control Alkaloids Terpenoids Steroids Tannins Glycosides.

In earlier Nandan Patel and Krishnappa (2017), studied, the Preliminary biochemicals screening from crude extract of Xylaria. carpophila fungus showed presence of alkaloids, tannins, flavonoids, sterols, glycosides, terpinoids and phenols.

HR-LCMS analysis. The results of HR-LCMS analysis of Claviceps purpurea extract resulted in the presence of some of the compounds (Table 2) and the chromatogram of the phytoconstituents is shown in Fig. 3. Among them, the compounds Aldicarb, Arecoline, Glimepiride, Gedunin and Pentobarbital are known for antibacterial properties Claviceps purpurea fungal secondary metabolites are very useful to drugs and can be directly extracted from the liquid broth using ethyl acetate as a solvent. In the present study, HR-LCMS analysis showed the presence of various compounds. Among them the compounds Aldicarb, Arecoline, Glimepiride, Gedunin and Pentobarbital are reported as worthy antibacterial agents (Cheloufi et al., 2014).

Fig. 3. HR-LCMS Chromatograph of Ethyl acetate extract of Claviceps purpurea.

Table 2: Molecular docking values of ethyl acetate extract of Claviceps purpurea fungal compounds obtained from LCMS analysis.

Antibacterial activity - Agar well diffusion assay. The antibacterial activity of Claviceps purpurea fungal extract was evaluated at the concentrations of 25, 50, 75 and 100 μg/m1 of DMSO and using different tested bacterial strains. In this test 100 μg/m1 concentrations showed significant antibacterial property against bacterial pathogenic strains like Pseudomonas aeruginosa (16.80±0.15), Escherichia coli (14.13±0.41), Staphylococcus aureus (13.37±0.27), Salmonella typhi (12.27±0.15), and Xanthomonas compestris (16.47±0.20), as compared to the standard drug ciprofloxacin.

In earlier studies S. aureus bacteremia is a significant cause of morbidity and mortality in neutropenic patients with cancer (Gonzalez et al., 2001). In our study, the metabolites of Claviceps purpurea exhibited significant inhibitory effect on both gram-positive Staphylococcus aureus, and gram-negative Salmonella typhi, Escherichia coli, Xanthomonas compestris and Pseudomonas aeruginosa strains which causes different disease symptoms. Previous studies of, The actinomycete isolate Streptomyces sp. VITBT7 was screened for antifungal and antibacterial activity on Sabaurauds Dextrose Agar (SDA) and Muller Hinton Agar (MHA) respectively. The cell free supernatant of the isolate exhibited antimicrobial activity against both Gram negative and Gram positive bacterial pathogens. The cell free supernatant also showed bactericidal activity with the inhibition zone of 37 mm against P. aeruginosa, 25 mm against K. pneumonia (Subashini and Kannabiran 2013).

Minimum Inhibitory Concentration (MIC). The MIC assay was performed by modified resazurin assay, the extract showed the highest inhibitory activity against Escherichia coli with a significant MIC value of 2.09±0.15×10-2. Inhibitions of bacterial strains are summarized in Table 3.

Table 3: Zone of inhibition and MIC values of ethyl acetate extract against tested bacterial strains.

Molecular docking studies

Toxicity prediction. Aldicarb, Arecoline, Glimepiride, Gedunin and Pentobarbital these five compounds showed pharmacokinetic properties and toxicity analysis properties identified by HR-LCMS as shown in Table 4. All the 5 compounds obey the Lipinski's ‘Rule of 5 limits better LogS values and were free from mutagenic tumorigenic, reproductive and irritant effect. In general, a poor solubility is associated with bad absorption and the aqueous solubility (Log S) of the compound which significantly affects its absorption and distribution characteristics. Based on the results from the Data Warrior, LogP, better LogS, and good drug score and less toxicity risk parameters are predicted as shown in the Table 4.

Table 4: In silico ADMET and drug-likeness prediction using data warrior.

In association with in vitro antimicrobial activity, it is useful to carry out in silico studies to predict the orientation and binding affinity at the active site of the receptor. The molecular docking of HR-LCMS identified ligand molecules are Aldicarb, Arecoline, Glimepiride, Gedunin and Pentobarbital. Among them, the compound gedunin exhibited better docking efficiency with DNA Gyrase. It forms three hydrogen bonds with amino acids His 1081, Gly 459 and Gly 458 in the active site of the target protein with bond length 2.94, 2.98 and 3.29 Å respectively, with the least binding affinity -6.3 and hence is considered as the best dock conformation (Table 5).

Fig. 4. 2D and 3D protein-ligand interaction DNA gyrase with the ligands aldicarb, gedunin, arecoline, pentobarbital and glimepiride.

Compound aldicarb forms one hydrogen bond with Ser438 amino acid with bond length of 3.03 Å. The compound arecoline forms two hydrogen bonds with the amino acids Asp437 with bond lengths 3.12 and 3.20Å and the compound Glimepiride forms three hydrogen bonding with Gly459, Arg1122 and: Ser1085 with bond length 2.80, 3.09 and3.24 Å respectively. The last compound pentobarbital forms two hydrogen bonds with amino acids Gly459 and Ser438 with bond length 3.11 and 3.19 Å respectively in this active pocket. However, all these docked molecules exhibited more hydrophobic interaction than the standard drug ciprofloxacin. The RMSD has often been used to measure the quality of reproduction of a known binding pose by molecules with ligands. All docked molecules have zero RMSD values as shown in the Table 5, Fig. 4. 

Table 5: Molecular docking values of ethyl acetate extract of Claviceps purpurea fungal compounds obtained from LCMS analysis.

Lokesh S.T., Sowmya H.V., Thippeswamy Basaiah and Ravikumar S.  (2025). Antibacterial and Molecular Docking Studies of Secondary Metabolites of Claviceps purpurea infected to Bajra (Pennisetum glaucum) Crop. Biological Forum, 17(5): 10-16.