Spines, stem and endophyte as three research materials
Kroenleinia grusonii spines have their own chemical compound profile. Heraiz and colleagues compared it with the composition of the stem and metabolites of a fungus isolated from inside the plant. In the 2026 publication, the cactus appears under its earlier name, Echinocactus grusonii. The material came from cultivation in Egypt and was collected in July 2023. Spines were separated from the stem by hand. Both materials were dried, ground and extracted separately with 80% methanol. This produced mixtures of substances soluble in this methanol–water system. [Heraiz et al. 2026, p. 2]
The fungus described as Aspergillus oryzae came from internal stem tissues. After surface disinfection of the plant, pieces of the interior were collected, and a pure culture was obtained through successive passages. A microorganism that colonises plant tissues in this way is called an endophyte. To produce analytical material, it was cultured for 21 days on a basmati rice medium. The culture was first extracted with ethyl acetate and the remaining material then with methanol. The two fractions obtained differed in preparation, composition and enzymatic activity. Their properties concern the laboratory culture, during which the fungus used an environment different from the cactus interior. [Heraiz et al. 2026, pp. 2–3]
How extract composition was determined
Ultra-high-performance liquid chromatography coupled with tandem mass spectrometry was used to separate the components. Chromatography separates the mixture, while the spectrometer records the masses of ions and their fragments. Comparing retention times and fragmentation patterns with published data allowed the authors to tentatively assign 27 metabolites to the spine extract and 14 to the ethyl acetate extract of the fungal culture. In the paper, these have the status of proposed identifications. This level of recognition provides a list of candidates for further purification and structural confirmation. [Heraiz et al. 2026, pp. 4–6]
The proposed spine components included alkaloids, including hordenine, flavonoids occurring as free molecules and glycosides, and phenolic acids. Glycosides contain a sugar moiety linked to the rest of the molecule. Derivatives of quercetin, kaempferol and isorhamnetin belonged to this group. The fungal extract profile included other groups of structures, including polyketides, alkaloids and lactones. Subsequent computer modelling concerned the structures proposed on the basis of these analytical data. [Heraiz et al. 2026, pp. 4–6, 7–10]
What content expressed as an equivalent means
In the Folin–Ciocalteu assay, the spine extract gave a result of 293,0 ± 3,2 mg of gallic acid equivalents per gram of dry extract. For the stem, the value was 145,9 ± 2,1 mg/g. The reagent reacts with components having reducing properties, and colour intensity is compared with a calibration curve. The result describes the analytical response of the mixture expressed in terms of a selected standard. The gram in the denominator here means dry extract obtained after solvent removal, rather than a gram of fresh plant. [Heraiz et al. 2026, Table 1, p. 4]
Similarly, alkaloid content was expressed as atropine equivalents. For the spine extract, the authors reported 14,46 ± 0,39 mg/g. Atropine served as the standard for converting the colour signal in this assay. Individual compounds were identified using a separate chromatographic method. Separating these two kinds of data distinguishes the magnitude of the response of an entire chemical group from information about a particular molecule. [Heraiz et al. 2026, pp. 3–4]
Inhibition of two enzymes
The biological part of the study comprised biochemical inhibition tests for acetylcholinesterase, abbreviated AChE, and the β-secretase BACE-1. AChE breaks down acetylcholine, while BACE-1 participates in amyloidogenic processing of amyloid precursor protein. Enzyme activity was measured using commercial screening kits. An IC50 was determined for each extract, the concentration causing half-inhibition of activity in the system used. A lower value meant that this effect was achieved with a smaller mass of test material per unit volume. [Heraiz et al. 2026, pp. 3, 6–8]
| Material tested | AChE | BACE-1 |
|---|---|---|
| Spines, methanolic extract | 0,362 ± 0,012 | 0,425 ± 0,014 |
| Stem, methanolic extract | 0,603 ± 0,019 | 0,939 ± 0,031 |
| A. oryzae culture, ethyl acetate extract | 0,255 ± 0,008 | 0,550 ± 0,018 |
| A. oryzae culture, subsequent methanolic extract | 3,180 ± 0,104 | 1,882 ± 0,062 |
| Donepezil, reference substance for AChE | 0,167 ± 0,006 | — |
| Curcumin, reference substance for BACE-1 | — | 0,350 ± 0,012 |
The spine extract inhibited both enzymes at lower concentrations than the stem extract. Among the extracts tested, the ethyl acetate fraction of the fungal culture showed the strongest AChE inhibition, while the lowest IC50 for BACE-1 was obtained for spines. The comparison also shows the importance of the extraction procedure. The subsequent methanolic extract of the fungal culture required higher concentrations than the ethyl acetate fraction. Each value refers to the entire mixture. Extract mass includes both components responsible for the effect and the other extracted substances. [Heraiz et al. 2026, Table 2, p. 8]
From chemical profile to a binding hypothesis
Molecular docking involved computational fitting of the proposed molecules to AChE and BACE-1 structures. For AChE, detailed analyses included terrusnolide A from the fungal profile and isorhamnetin rhamnoside from the spine profile. Versicolactone B and quercetin gentiobioside were selected for BACE-1. The model indicated possible contacts between the molecule and protein, including hydrogen bonds and interactions of aromatic rings. Docking scores served to compare the structures considered within the model used. [Heraiz et al. 2026, pp. 7–10]
Molecular dynamics simulations then tracked changes in the positions of the protein and bound molecule and the persistence of contacts. The authors observed that selected ligands remained in the modelled binding pockets. The result describes the behaviour of the complex under the computational assumptions. Experimental IC50 values, meanwhile, were determined for extracts. Combining the two parts of the study identifies candidates for purification, verification of identity and separate measurement of enzyme activity. [Heraiz et al. 2026, pp. 9–13]
The level of biological activity established
The study demonstrated inhibition of AChE and BACE-1 by the prepared mixtures in cell-free tests. The publication title refers to neuroprotection, but the endpoint of these experiments was enzyme activity. Neuronal survival, glial-cell responses and the course of disease in an organism remain separate levels of investigation. Similarly, the ability of components to penetrate the brain requires measurements that the described enzyme assays and simulations do not provide. [Heraiz et al. 2026, pp. 2–3, 6–14]
The next stage involves establishing which chemically confirmed components of the spines and endophyte culture account for the observed inhibition and how their activity changes after the mixture is separated. Comparing purified substances with active fractions would allow the contributions of individual molecules to be tested. The endophyte studied is also a separate source of material whose production can be analysed under controlled culture conditions. [Heraiz et al. 2026, pp. 4–14]
Based on the full text of the publication. Table data were checked against the image of the source PDF.
Therapeutic potential of Echinocactus grusonii and its associated endophytic fungus Aspergillus oryzae in neuroprotection: in vitro and in silico investigations
Heraiz A. A., Othman A., Farouk A., Hegazy M. M., Mohammed A. E. S. I., El-Hela A. A., Therapeutic potential of Echinocactus grusonii and its associated endophytic fungus Aspergillus oryzae in neuroprotection: in vitro and in silico investigations, Scientific Reports, 2026, 16(1):23556, doi: 10.1038/s41598-026-62565-5
A comparison of spine and stem extracts of Echinocactus grusonii, now Kroenleinia grusonii, with culture extracts of the endophytic Aspergillus oryzae isolated from inside the stem. The chemical analysis includes assays of compound groups and preliminary metabolite identifications using UPLC-MS/MS. The authors measure inhibition of AChE and BACE-1 by the mixtures, and analyse proposed structures of selected components using docking and molecular dynamics. The study includes neither a neuronal model nor a clinical experiment.