Research Article

Extraction and Production of Ergot Alkaloids Produced by Fungal Consortium

Zohaib Anjum1*, Memuna Ghafoor Shahid1, Ikram-ul-Haq2, Awais Anjum1, Mehak Iftikhar1 and Nazish Mazhar Ali3

1Department of Botany, Government College University, Lahore, Pakistan; 2Department of Biotechnology, Government College University, Lahore, Pakistan; 3Department of Zoology, Government College University, Lahore, Pakistan.

Received | July 24, 2025; Accepted | September 17, 2025; Published | September 27, 2025

*Correspondence | Zohaib Anjum, Department of Botany, Government College University, Lahore, Pakistan; Email: [email protected]

Citation | Anjum, Z., M.G. Shahid, I. Haq, A. Anjum, M. Iftikhar and N.M. Ali. 2025. Extraction and production of ergot alkaloids produced by fungal consortium. Pakistan Journal of Weed Science Research, 31(3): 190-195.

DOI | https://dx.doi.org/10.17582/journal.pjwsr/2025/31.3.190.195

Keywords | Fungal consortium, Ergot Alkaloids, OFAT, Extraction, Production, Parameter

Copyright: 2025 by the authors. Licensee ResearchersLinks Ltd, England, UK.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

Introduction

Ergot alkaloids are a type of secondary metabolite produced primarily by the fungal genus Claviceps that parasitize cereal crops such as rye and grasses, posing both toxicological risks and pharmaceutical benefits (Van-Urk, 1992). These compounds, named after the Old French term “argot” meaning cock spur, have been used for a long time to treat migraines and other biological processes, postpartum hemorrhage, and conditions requiring oxytocic effects (Flieger et al., 1997).

Structurally, ergot alkaloids are classified into three main categories: Peptides (ergopeptines), lysergic acid amides, and clavinet (Agriopoulou, 2021). The synthesis of these alkaloids involves complex biochemical processes influenced by fungal species, host plants, and microbial interactions in their environment (Steel and Torrie, 1996). While Claviceps purpurea has been traditionally connected with ergotism outbreaks, recent study underlines the importance of fungal consortia in boosting ergot alkaloid production through mutualistic and antagonistic interactions (Fabian et al., 2018). These alkaloids, although mycotoxins with neurotoxic effects, have considerable therapeutic potential, particularly in the treatment of CNS illnesses such as Parkinson’s disease and migraines (Florea et al., 2017; Shahid and Nadeem, 2015). Their mechanism of action involves interactions with adrenergic, serotonin, and dopamine receptors in the nervous and cardiovascular systems (Gerhards et al., 2014).

The classic method of obtaining ergot alkaloids is cultivating Claviceps fungi on sensitive host plants and extracting the ergot sclerotia; however, this process is labor-intensive and subject to environmental variables, resulting in uneven yields (Keller et al., 2005). Biotechnology advancements now focus on optimizing fungal consortia to increase production efficiency (Hulvová et al., 2013). Industrial biosynthesis employs a variety of fermentation processes, including surface culture, submerged, and solid-state fermentation (Řičicová et al., 1982).

Thin Layer Chromatography (TLC) and Liquid Chromatography-Mass Spectrometry (LC-MS) are two analytical techniques used to identify and quantify these chemicals in grains, grasses, and medicinal formulations (Faiq et al., 2025). Given their pharmacological importance, research into optimizing ergot alkaloid synthesis remains critical for medical and industrial applications, necessitating a thorough understanding of fungal consortia dynamics and environmental influences in order to maximize production and therapeutic potential (Shahid and Nadeem, 2015).

Materials and Methods

Fungal culture procurement

Fungal consortium including Penicillium digitatum, Trichoderma viridii and Mucor mucedo, was produced from the Cell Culture Insight Laboratory, Department of Botany, Government Collage University, Lahore Pakistan. This consortium was chosen due to the fungi’s established role as producers of extracellular enzymes and secondary metabolites that hold biotechnological significance. Penicillium digitatum is an efficient producer of α-amylase, Trichoderma viridii exhibits strong cellulolytic activity, and Mucor mucedo contributes to enhanced metabolite diversity.

Maintenance of fungal culture

Penicillium digitatum, Trichoderma viride, and Mucor mucedo fungal cultures were maintained by creating MEA slants and then inoculating fungal strains into them. MEA medium were prepared, adjusted to pH 7.0, sealed, and autoclaved at 121°C for 20 minutes.

Preparation of inoculum

Spore suspension was prepared by scratching fungal growth from slants, adding 5ml of distilled water, vortexing to separate mycelium, and yielding roughly 100 spores per milliliter.

Fermentation technique

In one liter of fermentation medium that contained sucrose, yeast extract, orange waste, peptone, CaCl2, KH2PO4, MgSO4, ZnSO4, and FeSO4, the fungal strain was cultivated. Adjusting the pH to 5 using 0.1N HCl and ammonia solution. To ascertain ergot alkaloid production, the medium was incubated at 25°C for 21 days following inoculation using the surface culture fermentation technique.

Optimization of fermentation parameters

The impact of pH, incubation period, and inoculum size on mycelium development and ergot alkaloid synthesis was investigated by varying pH (3.0-8.0), incubation periods (3-13 days), and inoculum sizes (5-30 ml). These investigations helped to discover the optimal conditions for optimum alkaloid synthesis.

Determination of ergot alkaloids

Ergot alkaloids were measured by separating the supernatant and mycelium, centrifuging, and incubating with Van Urk reagent at 590 nm to quantify optical density (OD) for extracellular and intracellular enzymes. The mycelial mass was dried, homogenized in methanol, and centrifuged to extract intracellular alkaloids, and the optical density was determined following incubation.

Ergot alkaloids assay

To determine the ergot alkaloid yield, several standard salt concentrations (40, 80, 120, 160, and 200g) were produced in sterilized double-distilled water with stock solutions and incubated with Van Urk reagent for 30 minutes at 37°C. The optical density (OD) at 590 nm was then measured with a spectrophotometer, using a blank of 1 mL clean water and 2 mL Van Urk reagent (Scott, 2007; Smith 1930).

Extraction of ergot alkaloid

It was carried out by shaking 30 mL of chloroform and 10 mL of enzyme extract in a separating funnel three times, then collecting the bottom layer for additional cleaning. Thin layer chromatography (TLC) was performed on Merck silica gel plates to compare the alkaloid content of samples with reference salts BCM and DMS, as well as samples prepared by chloroform extraction, followed by analysis with various mobile phases, and alkaloids detected using Van Urk reagent and UV light at 254nm, with Rf values calculated (Králová et al., 2021).

Statistical analysis

To reach a final conclusion, various parameters were statistically evaluated to record data using standard deviation and standard error (Steel and Torrie, 1996).

Results and Discussion

The optimization of fermentation media for ergot alkaloid synthesis revealed that M2 medium provided the largest quantity in both mycelium (0.673 mg/ml) and supernatant (0.448 mg/ml), compared to M1, which had lower yields (mycelium: 0.629 mg/ml, supernatant: 0.324 mg/ml) as shown in Table 1.

 

Table 1: Fungal consortium’s selection of fermentation medium for Ergot alkaloids production.

S. No.	Different fermentation medium	Supernatant (mg/ml)	Mycelium (mg/ml)
1	M1	0.324±0.001	0.629±0.001
2	M2	0.448±0.001	0.673±0.001

 

Effect of pH and incubation period on ergot alkaloids production

Ergot alkaloid yield was maximum at pH 5 (2.90 mg/ml) in the supernatant, and lowest at pH 3 (1.01 mg/ml), with no mycelium development. The highest yield (2.92 mg/ml) was achieved after 9 days of incubation, whereas the lowest yield (0.19 mg/ml) was seen after 3 days (Figure 1).

Effect of peptone and CaCl2 on ergot alkaloids production

The highest ergot alkaloid production was observed at 20g peptone (2.95 mg/ml) in the supernatant, while the lowest was at 5g peptone (0.300 mg/ml), with the highest mycelium growth at 5g peptone (2.91 mg/ml). For CaCl₂, the maximum production was at 0.75g (1.623 mg/ml) in the supernatant, and the highest mycelium growth was at 1.5g (0.312 mg/ml) (Figure 2).

 

 

Effect of carbon, nitrogen sources and KH2PO4 on ergot alkaloids production

Fructose produced 3.21 mg/ml of ergot alkaloid in mycelium, while maltose yielded the least (0.379 mg/ml) and peptone produced the highest nitrogen-based output (2.95 mg/ml). The maximum outputs were obtained at KH₂PO₄ concentrations of 0.5g in supernatant (1.526 mg/ml) and 1.5g in mycelium (1.618 mg/ml) (Figure 3).

 

Extraction of alkaloids

Ergot alkaloids were extracted from a one-liter fermenter, giving 1.22±0.01 mg/mL. The alkaloids were then examined using thin layer chromatography (TLC), with mobile phase F selected for separation, yielding an Rf value of 0.81 for the supernatant, which was validated by Van Urk reagent as shown in Table 2.

 

Table 2: Rf values of supernatant or mycelium produced by fungal consortium.

Mobile phase	Supernatant	Mycelium	Rf value	Expected ergot alkaloids
A	-	-	-	-
B	-	-	-	-
C	-	-	-	-
D	-	-	-	-
E	-	-	-	-
F	+	-	0.81	Ergocriptine

 

Discussion

Ergot alkaloids are essential to the pharmaceutical industry since they are used to make uterotonics, prolactin inhibitors, anti-Parkinson pharmaceuticals, anti-migraine therapies, and other medicinal substances Hulvova et al (2013). Table 1 shows the maximum levels of ergot alkaloids production and mycelial development. The current study looked at different pH values (Figure 1), and it discovered that the generation of ergot alkaloids was best at pH 5, producing 2.90 mg/ml. These findings are consistent with those of Shahid and Nadeem (2015). The use of a fungal consortium for ergot alkaloid production has not previously been documented in the literature, and this study highlights this as a novel aspect of the current work. The consortium strategy was chosen because the interaction of various fungi can increase metabolite diversity and overall production via complementary enzymatic activity and biosynthetic routes. Claviceps purpurea, a traditional ergot alkaloid generator, was not included in this study due to its pathogenic nature, slow growth, and strict host-dependent needs, making it inappropriate for laboratory-scale investigation. As a result, the chosen consortium (Penicillium digitatum, Trichoderma viridii, and Mucor mucedo) presents a safe and unique alternative for efficient Ergot alkaloid production.

The effects of inoculum size and incubation length were also investigated (Figure 1). An inoculum size of 30 ml and a nine-day incubation time produced the highest ergot alkaloid production. According to Taber and Vining (1958), Smith (1930), Shahid et al. (2017), a normal water-suspended source might provide significant amounts of ergot alkaloids with an inoculum of 1-2 milliliters. Additionally, peptone concentration was optimized; the highest yield of 2.95 mg/ml was obtained with 20 g of peptone (Figure 2), which is in line with earlier research on the manufacture of clavine alkaloids (Liu et al., 2022). Our results are consistent with those of Leadmon et al. (2022), who reported that the generation of chaetominine was gradually reduced by raising the CaCl2 concentration, with the maximum yield being found at 1.5 g of CaCl2 concentration, or 0.312mg/ml, respectively (Figure 2).

A variety of substrates were examined in order to identify the best carbon source for optimizing the generation of ergot alkaloids (Figure 3). In mycelium, fructose produced the largest alkaloid output (3.21 mg/ml), whereas maltose produced the lowest yield (0.379 mg/ml). After more fructose concentration adjustment, it was found that a 25 g fructose concentration resulted in the maximum alkaloid yield in the supernatant (2.91 mg/ml), whereas 10 g fructose provided the lowest yield (0.0005 mg/ml) (Figure 3). Instead of promoting excessive fungal growth, fructose increases alkaloid biosynthesis, and when combined with glucose, it inhibits alkaloid synthesis according to Brar et al. (1968).

Peptone was found to be an essential supply of nitrogen for the formation of the inoculum, and different doses were examined in order to maximize the yield of alkaloids. According to recent research on the function of peptone in clavine alkaloid synthesis and fungal development (Leadmon et al., 2022), the maximum yield (2.95 mg/ml) was obtained with 20 g of peptone (Figure 3) (Liu et al., 2022; Won et al., 2022). The ideal KH₂PO₄ concentration was also found to be 1.5 g, which results in 1.618 mg/ml and is in line with other studies (Fabian et al., 2018). There has also been a great deal of research on the effects of varying KH₂PO₄ concentrations on the formation of ergot alkaloids (Figure 3) (Tudzynski et al., 2021).

The chloroform extraction procedure was used in this investigation to partially purify the ergot alkaloids (Table 2). One liter of optimized fermenter culture produced 1.26 mg/ml of pure alkaloids, which is consistent with Shahid et al. (2017) findings. Our results also align with prior studies that use the G mobile phase (chloroform 140 ml: Propanediol 55 ml: water 15 ml) in an Aspergillus niger extract to get the highest Rf value (0.99) (Shahid and Nadeem, 2015). Additionally, for metabolite separation, a mobile phase consisting of 1.6 ml acetic acid, 20 ml acetone, and 78.4 ml chloroform was investigated (Taber and Vining, 1958).

Conclusions

This study found that the best carbon source for increasing the formation of ergot alkaloids was fructose (3.21mg/ml in mycelium), whereas the best nitrogen source was peptone (2.95mg/ml in supernatant). The best yields were obtained at 0.5g KH₂PO₄ (1.526 mg/ml) and 1.5g KH₂PO₄ (1.618 mg/ml). With an Rf value of 0.81, TLC analysis verified the existence of ergot alkaloids, underscoring the study’s importance for medicinal and biotechnological uses. Ergot alkaloids are important in pharmacology and are used to treat prolactin as well as a number of illnesses, including Alzheimer’s disease and migraines.

Acknowledgement

We are thankful to the Department of Botany, overnment College University Lahore, Pakistan, for roviding all the technical facilities and chemicals to omplete this research.

Novelty Statement

The work on ergot alkaloids production and dentification is very limited here in Pakistan. Our esearch group synthesized these pharmaceutically and herapeutically important alkaloids through the use of ungal consortium and we produced a novel alkaloid amed ergocrptine in our research.

Author’s Contribution

Zohaib Anjum: Main Research conducted, experiments, analysis, draft.

Dr. Memuna Ghafoor Shahid: Supervisor, idea, experiments, analysis, editing of draft.

Ikram-ul-Haq: Editing in draft and analysis.

Awais Anjum: Statistical Analysis and draft editing.

Nazish Mazhar Ali: Editor and Analysis.

Generative AI and AI-assisted technology statement

No AI assisted technology was used in generation of this draft.

Conflict of interest

The authors have declared no conflict of interest.

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