LC–HRMS metabolomic profiling of Anisosciadium lanatum and Its antioxidant and phytotoxic potential

Abstract

Our study aims to explore the antioxidant and allelopathic effects of ethanolic and aqueous extracts of Anisosciadium lanatum. Antioxidant assays were assessed using 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2’-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid (ABTS), and β–carotene bleaching methods. The phytotoxic effect was analyzed against seeds of Triticum aestivum L., Raphanus sativus L., and Lens culinaris L. The in silico study was performed using the Auto Dock 4.2 software package. The aqueous extract exhibited the highest antioxidant capacity with IC₅₀ values of 13 µg/mL, 8 µg/mL, and 50 µg/mL for DPPH, ABTS+, and β–carotene bleaching, respectively. This sample had the highest levels of total flavonoids, total phenolics, and total tannin content. The aqueous extract significantly stimulated wheat stems and roots at low concentrations, while it showed significant inhibition at high concentrations. Using Liquid Chromatography-High Resolution Mass Spectrometry, sixty compounds were tentatively identified in the investigated species Anisosciadium lanatum, and based on currently available literature, these compounds have not been previously reported in the Anisosciadium genus. The most abundant compounds based on relative peak areas in the ethanolic sample were 2’’,3’’-Di-O-p-coumaroylafzelin, kaempferol 3-O-β-D-galactoside, and procyanidin B6, whereas luteolin 4’-O-glucoside, myricetin 7-rhamnoside, and 3-methylellagic acid 8-rhamnoside showed the highest relative abundances in the aqueous extract. Procyanidin B6 demonstrated the highest binding affinity toward 4-hydroxyphenylpyruvate dioxygenase, showing a binding energy of –10.6 kcal/mol, while myricetin 7-rhamnoside (–5.4 kcal/mol) exhibited the most favorable binding affinity toward Human peroxiredoxin 5, revealing its prospectivity as a potent inhibitor. Our findings confirm that A. lanatum is a rich source of bioactive molecules with significant antioxidant and allelopathic effects.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-026-49823-2.

Introduction

In recent decades, the topic of natural antioxidants has gained significant attention in the food industry, human nutrition, and medicine. Nutritional researchers have frequently noted that the harmful effects of diseases associated with reactive oxygen species (ROS) can be mitigated by incorporating antioxidant compounds into human diets. The production of ROS has been linked to immediate DNA damage, lipid peroxidation, and protein carbonylation, contributing to a range of disorders and chronic health conditions, including cancer, neurological diseases, aging, and cardiovascular issues¹. Conversely, the increased use of synthetic herbicides has raised concerns about their impact on human health and the environment. This has sparked research into natural compounds and alternative approaches for efficient weed management in agricultural settings². Among these strategies, allelopathy has emerged as a viable pest control method, offering sustainable and ecologically integrated weed management solutions³. To reduce reliance on synthetic pesticides, collaborative efforts have been made to discover plant-derived products that are safe and eco-friendly⁴. The edible plant A. lanatum Boiss belongs to the Apiaceae family, comprising 434 genera and 3780 species. It is distributed worldwide^5,6, particularly in temperate areas. Several well-known herbs and vegetables are part of this family, including coriander/cilantro, dill, celery, parsnip, carrot, angelica, fennel, cumin, caraway, and parsley⁷. Many Apiaceae species are edible and exhibit various biological and pharmacological activities, including anti-inflammatory, anti-tumor, hepatoprotective, antibacterial, and cyclooxygenase inhibitory effects⁵. Additionally, numerous species serve as vital sources of essential oils⁸. The genus Anisosciadium is endemic to Southwest Asia and comprises three species: A. lanatum Boiss, A. isosciadium Bornm., and A. orientale DC.⁹. A. lanatum is a perennial plant featuring segmented, cleft, and webbed base leaves, characterized by compound-type inflorescences. Its small flowers have pinkish-white petals and spiny head bracts. The fruits of each secondary navel aggregate form before maturity but later separate into spiny units^10,11. A. lanatum is native to and widely distributed throughout the Arabian Peninsula, including Saudi Arabia^12,13. In traditional medicine, Bedouins have utilized A. lanatum as a local medicinal herb; a water extract from the dried aerial parts, including flowers, fruits, stems, and leaves, is used to treat skin sores and boils¹⁴. Additionally, for Bedouin children, young green leaves are an acceptable and refreshing herb^15,16. Furthermore, A. lanatum has demonstrated veterinary significance, as extracts from its leaves and shoots have been used to treat skin conditions in goats and sheep¹². Previous studies indicated that this plant exhibited anti-proliferative, lipoxygenase, and antimutagenic activities^16,17. To date, two separate studies have documented the antioxidant properties of A. lanatum. The ABTS method was used to assess antioxidant activity in plant samples collected in Riyadh in one study¹⁷, while another study used the DPPH assay on plant samples from the Eastern Region of Saudi Arabia¹⁶. The β-carotene bleaching assay has not been used to evaluate the antioxidant properties of A. lanatum, and the ethanolic extract of this species has not been tested. Besides, only two compounds were isolated from this species (anisosciadone and stigmasterol)¹⁸. Previous studies were limited to qualitative chemical analyses, mainly focusing on the determination of total phenolic and flavonoid contents, without detailed identification of individual metabolites^16,17. The analysis of A. lanatum metabolites by LC–HRMS has not yet been reported. The current research indicates that additional studies are needed, as scientists have conducted only a few tests using different methods to extract antioxidants from various plant sources. The genus Anisosciadium lacks research investigating the allelopathic properties of any of its species. The research investigates A. lanatum specimens collected from the Hail region, which is more than 800 kilometers from all previously documented sites. The study uses three in vitro assays to evaluate A. lanatum’s antioxidant properties and tests its phytotoxic effects on wheat, lentil, and radish seeds. The experimental results are supported by in silico ADME and molecular docking studies of 4-hydroxyphenylpyruvate dioxygenase and human peroxiredoxin 5, which reveal possible molecular binding mechanisms that explain the observed biological effects. The combined research method yields new insights into A. lanatum’s biological properties, benefiting both agricultural sustainability and industrial production.

Materials and methods

Chemicals and reagents

Pure chloroform (CHCl₃), 99.99% Ethanol (C₂H₆O), distilled water, sodium hypochlorite (1%) (NaOCl), deionized water, Folin–Ciocalteu reagent (FC reagent), Na₂CO₃ (sodium carbonate solution), gallic acid, aluminum chloride (AlCl₃-6H₂O), quercetin, vanillin, sulphuric acid (H₂SO₄), catechin, 2,2’-azinobis (3-ethyl-benzothiazoline-6-sulfonic acid (ABTS), 1,1-Diphenyl-2-picrylhydrazyl (DPPH^.), β-carotene, linoleic acid and ascorbic acid, were bought from Sigma–Aldrich (Germany).

Plant material and extraction process

A. lanatum aerial plant material (1.5 Kg) was collected from Farms of Simira governorate, South Hail, Kingdom of Saudi Arabia, in March 2023, after obtaining verbal authorization from the landowners. The geographical coordination of Simira governorate is 26° 21’ 4” North, 41° 43’ 30” East. A. lanatum used in this study was collected in accordance with applicable institutional, national, and international guidelines and regulations. The plant was formally identified by Dr. Belsem Marzouk (Faculty of Pharmacy, University of Monastir) and authenticated as A. lanatum, belonging to the family Apiaceae, based on macroscopic and microscopic examination. A voucher specimen (A.l 83) was put in the herbarium of the laboratory of biology (HBL) in the College of Sciences (Simira branch –Hail University). The plant aerial parts underwent shade drying at room temperature before being blended into a powdered form using an electric blender. The powdered material underwent 600 g maceration treatment with progressive solvent polarity escalation. The plant powder required 2.5 L of chloroform for 72 hours of maceration at room temperature inside a sealed container, which needed periodic stirring. The mixture required filtration through Whatman No. 1 filter paper, and the residue was air-dried to remove residual solvent. The dried residue underwent ethanol maceration with 2.5 L of solvent for 72 hours while maintaining the same conditions before filtration occurred. The ethanolic extract underwent concentration through reduced pressure at 40°C while using a rotary evaporator. The final residue required 2.5 L of distilled water for 72 hours of room temperature extraction before filtration occurred. The aqueous extract underwent freezing followed by lyophilization to achieve dryness. The ethanolic sample yielded 2.0 g of dry residue after removing the solvent which represented 0.33% (w/w) of the starting dry plant material. The aqueous extract produced 3.5 g which represented an extraction yield of 0.58% (w/w). The extraction yields were expressed as percentages relative to the dry weight of the dried plant material. The chloroform extract was not included in subsequent biological or chemical investigations; therefore, its extraction yield was not reported in the manuscript¹⁹.

Chemical analysis

Quantification of total phenolic content

The total phenolic content of A. lanatum samples was determined according to the previously published study¹⁹. 100 μL of the extract (1000 μg/mL) was mixed with 750 μL of Folin-Ciocalteu reagent. This mixture was allowed to stand at room temperature for 5 minutes. After that, 750 μL of Na₂CO₃ (sodium carbonate) was added. The reaction was mixed entirely and allowed to stand for 90 min at 25°C in the dark. The optical density of the sample solutions was measured at 725 nm using a UV-visible spectrophotometer. The experiments were performed in three replicates. The TPC of extracts was expressed as mg gallic acid equivalents (GAE) per g of extract. The calibration curve was prepared using a gallic acid stock solution.

Quantification of total flavonoid content

The total flavonoid content of different samples (1000 μg/mL) was determined according to the previously published study¹⁹. 1500 μL of extracts were combined with an equal volume of a 2% AlCl₃-6H₂O solution. The mixture was hardly agitated. After 10 minutes of incubation, the absorbance was measured at 367 nm using a UV spectrophotometer. The total flavonoid content was expressed as μg quercetin/mg dry weight (mg QE/g DW) by the calibration curve of quercetin. The experiments were performed in three replicates. We used a calibration curve ranging from 0 to 50 μg/mL (R² = 0.99).

Quantification of total tannin content

The total tannin content was determined using the modified vanillin assay described by Hlila et al. (2015)¹⁹. 3000 μL of 4% methanol vanillin solution and 1500 μL of concentrated H₂SO₄ were added to 50 µL of a suitably diluted extract (1000 μg/mL). After 15 minutes, the absorbance was measured at 500 nm against methanol as a blank using a UV spectrophotometer. The content of total tannin is expressed as mg catechin/g dry weight (mg CE/g DW). The experiments were performed in three replicates. The calibration curve range was 0–400 µg/mL (R²= 0.99).

Antioxidant activity

DPPH radical scavenging activity

The DPPH radical scavenging activity was measured according to a previously published study²⁰. This method is a screening test for evaluating the antioxidant activity of samples. 50 µL of the extract solution were added to 200 µL of the newly prepared solution of 4*10^–3 % DPPH in MeOH. After 30 min of incubation in the dark at 37°C, absorbance was recorded at 517 nm using a UV spectrophotometer. The following concentrations (2.4, 4.8, 9, 19, 39, 78, 156, 312, 625, 1250, 2500, and 5000 μg/mL) were used in this test. The experiments were performed in three replicates. This test was manifested as IC₅₀ in μg/mL.

ABTS^.+ method

The antioxidant activity was tested in vitro. It is based on scavenging ABTS⁺ by antioxidants in the plant extract, resulting in a spectrophotometric decrease in absorbance at 734 nm using a UV spectrophotometer. We dissolved the extracts separately in ethanol to obtain these concentrations (2.4, 4.8, 9, 19, 39, 78, 156, 312, 625, 1250, 2500, and 5000 μg/mL). The experimental protocol details were provided in our earlier research study¹⁹. The experiments were performed in three replicates. This test was manifested as IC₅₀ in μg/mL.

β-Carotene bleaching inhibition activity

This assay was defined following the method detailed in our previous work, with some modifications¹⁹. 2000 µL of β-carotene solution was added to 20 µL of linoleic acid and 200 µL of Tween 20. Then, 50000 μL of distilled water was added to the dried mixture to form a β-carotene-linoleic acid emulsion. 5000 µL of emulsion was added to 500 µL of samples at various concentrations: 2.4, 4.8, 9, 19, 39, 78, 156, 312, 625, 1250, 2500, and 5000 μg/mL. The mixtures were incubated in a water bath at 50°C for 2 hours, and the absorption of the reaction mixtures was read at 470 nm using a UV spectrophotometer. The experiments were performed in three replicates. The antioxidant activity (AA) of the extracts was calculated utilizing the following equation: AA% = (β-carotene content after 2 h / initial β-carotene content) * 100¹⁹ .The extract concentration resulting in 50% inhibition (IC₅₀) was determined by plotting the inhibition percentage against sample concentration.

Phytotoxic activity

The protocol for assessing phytotoxic activity was determined according to our previous work²¹, with some modifications. The different extracts prepared were placed in Petri dishes lined with filter paper after dissolving in water. Five final concentrations were prepared for the allelopathic assay: 625 μg/mL, 1250 μg/mL, 2500 μg/mL, 5000 μg/mL, and 10000 μg/mL. The pH of each treatment solution was measured using a calibrated pH meter, adjusted to 6.8 ± 0.2 with dilute HCl or NaOH when necessary, and rechecked at the beginning and end of the experiment to ensure stability. Seedlings watered with distilled water served as the control. All samples were tested on T. aestivum L. (wheat), R. sativus L. (radish), and L. culinaris L. (lentil) seeds. The seeds were surface sterilized with 525 µg/mL sodium hypochlorite for 2 min.

The seeds were rinsed four times with distilled water, imbibed in it at 22°C for 12 hours, and carefully blotted dry using a folded paper towel. Ten imbibed seeds of the target species were separately placed on filter paper in Petri dishes. They were covered and incubated in a controlled-environment growth chamber at 25 ± 2°C, 55 ± 5% relative humidity, and a 10:14 light: dark photoperiod. Light was supplied at approximately 180 µmol.m^–2.s^–1 photosynthetic photon flux density (PPFD). Germination was assessed by counting the number of germinated seeds (seeds where a radicle emerges) at 24-hour intervals over 7 days. The lengths of the roots, epicotyls, or coleoptiles of the investigated species were measured on all seedlings in each Petri dish on day 7 after seeds were placed on the medium. The control for this experiment consisted of seeds imbibed with distilled water. Data from three replicates were expressed as a percentage of the control. The inhibition percentage was calculated using the following equations:

1) Germination Percentage (GP) = (germinated seeds / total number of seeds) * 100.

2) Inhibition Percentage of Roots and Epicotyls or Coleoptiles length (IPR, IPE, and IPC) = [(control length − extract length) / control length] * 100. Here, “extract” refers to the measurement parameter in the presence of the sample, while the “control” is measured in the presence of distilled water.

Phytochemical profile of A. lanatum extracts

The phytochemistry of A. lanatum ethanol and water extracts was analysed using a UHPLC-PDA-High-Resolution Mass Spectrometer (1290 Infinity UHPLC system; Agilent Technologies®, Santa Clara, CA, USA). The liquid chromatographic system included a HiP sampler, a binary gradient solvent pump, a column compartment, and a quadrupole time-of-flight mass spectrometer (MS Q-TOF) equipped with a dual Agilent Jet Stream Electrospray (AJS ES) ion source. A sample volume of 3 µL was injected into the system and separated on an SB-C18 column (2.1 mm × 50 mm, 1.8 µm particle size) maintained at 40 °C. Solvent A consisted of 0.1% formic acid in deionized water, while solvent B was acetonitrile, with a flow rate set at 0.300 mL min^–1. Mass spectrometric detection was performed in positive-ion MS Q-TOF mode using AutoMS² acquisition over m/z 120–1200. Putative compound annotation was based on accurate mass measurements, agreement of isotopic patterns, and MS/MS fragmentation patterns acquired with a medium isolation width (~4 amu) and ramped collision energy. Accordingly, all HR-LCMS compound assignments are reported as putatively annotated metabolites corresponding to the Metabolomics Standards Initiative (MSI) Level 2. The main tools used for phytochemical annotation included ChemSpider and PubChem databases. The mass spectrometer was routinely calibrated before analysis, and analyses were carried out under identical conditions to ensure repeatability²².

In silico study

The in Silico study was detected using the same protocol as described in our previous study, with some additions²¹. Indeed, the crystal structures of Arabidopsis thaliana HPPD complexed with NTBC (PDB: 6J63) and Human peroxiredoxin 5 (PDB: 1HD2) were downloaded from the RSCB protein data bank (https://www.rcsb.org/). The ligands are prepared as follows: the molecules to be tested are drawn and then optimized using the ACD (3D viewer) software (http://www.filefacts.com/acd3d-viewer-freeware-info). These ligands are then saved with the extension (.mol) and opened using Discovery Studio 2017R2 software (https://www.3dsbiovia.com/products/collaborative-science/bio via-discovery-studio/) and saved with the extension (.pdb). Regarding the preparation of the target enzyme, the enzyme is opened using SPDBV software, the receptor is cleaned of water molecules and co-complexed ligands, and then the missing hydrogens and Gasteiger charges were added to the system during the preparation of the receptor input file. AutoDock Tools were used to prepare all ligands and proteins files (PDBQT). Then, after preparing the ligands (.pdb) and receptors (.pdb), the active site coordinates of each target enzyme were fixed: X, Y, and Z coordinates, which in our cases are as follows: the center X=30.157, Y=-22.959, and Z=4.505 for the pdb enzyme 6j63, and the center X=7.023, Y=41.721, and Z=34.408 for the pdb enzyme 1hd2, using GID boxes (size_x=20, size_y=20, and size_z=20). Once the calculation was complete, the binding energy values were exhibited. We opened the docking complex using PMV (an interface linked to Autodock software) to save the relevant position with the .pdb extension. The visualization and analysis of interactions were executed using Discovery Studio 2017R2 and PyMOL 0.99rc6 (Open-Source).

Statistical analysis

The results appeared as mean values with standard error (SE) measurements from at least three independent replicates for each extract. The research team conducted their statistical analysis through IBM SPSS Statistics version 22 (IBM Corp., Armonk, NY, USA). The Shapiro–Wilk test and Levene’s test were used to verify normality and homogeneity of variances in the data before starting the analysis. The analysis of variance (ANOVA) procedure was applied because the assumptions of normality and homoscedasticity were met (p > 0.05). The researchers performed Duncan’s multiple range test to compare means between groups at a significance level of p < 0.05 after they detected substantial differences between the groups. IC₅₀ values for the DPPH, ABTS, and β-carotene/linoleic acid methods were calculated from dose–response curves and expressed with their corresponding 95% confidence intervals (CI).

Results

Chemical analysis

The A. lanatum water extract showed the highest phenolic content (90.36 µg GAE/mg), followed by the ethanol extract (76.6 µg GAE/mg) (Fig 1a). The water fraction also presented the highest flavonoid content (6.95 μg QE/mg), followed by the ethanol extract (5.02 μg QE/mg) (Fig 2b). The water extract showed the highest tannin content (80.32 µg catechin/mg) compared to the ethanol extract (63.1 µg catechin/mg) (Fig 1c).

Fig. 1.

Chemical analysis of A. lanatum ethanol and water extracts; total phenolic content (a), total flavonoid content (b), total tannins content (c). Ale: A. lanatum ethanol extract; Alw: A. lanatum water extract; µg GAE/mg: Microgram gallic acid equivalent per milligram; µg QE/mg: Microgram quercetin equivalent per milligram; µg CE/mg: Microgram catechin equivalent per milligram mg. The letters (a – b) on the bars represent significant differences (p < 0.05).

Fig. 2.

Inhibition or stimulation percentage of epicotyls or coleoptiles length of (a) wheat, (b) Radish, and (c) lentil in the presence of A. lanatum ethanolic (Ale) and A. lanatum water extracts (Alw). Negative inhibition values represent growth stimulation. Each bar represents the average ± SE with 3 repetitions (n = 30). The letters (a–j) on the bars imply significant differences (p < 0.05).

Antioxidant activity

DPPH radical scavenging activity

The free radical scavenging possibility of the ethanolic and water samples of A. lanatum was established through the change in optical density caused by the reduction of the DPPH radical, with results expressed in Table 1 as IC₅₀ (µg/mL). The water extract showed the best results, with an IC₅₀ of 13.0 µg/mL (95% CI: 12.2-13.4 µg/mL), followed by the ethanol fraction, with an IC₅₀ of 20.0 µg/mL (95% CI: 19.4-20.8 µg/mL). The antioxidant study of the tested samples, compared with ascorbic acid as a standard, with an IC₅₀ of 11.0 µg/mL (95% CI: 10.8-11.7 µg/mL), showed similar findings.

Table 1.

IC₅₀ (μg/mL) of Ale and Alw extracts compared to ascorbic acid with 95% confidence intervals.

Assay	Ale IC50 (μg/mL)	Alw IC50 (μg/mL)	Ascorbic acid IC50 (μg/mL)
DPPH	20 (19.4-20.8)	13 (12.2-13.4)	11 (10.8-11.7)
ABTS	21 (20.3-21.8)	8 (7.4-8.6)	15 (14.3-15.7)
β-carotene	63 (61.2-64.7)	50 (49.3-50.4)	48 (47.2-50.2)

DPPH: 1,1-Diphenyl-2-picrylhydrazyl, ABTS: 2,2’-azinobis (3-ethyl-benzothiazoline-6-sulfonic acid. Ale: A. lanatum ethanol extract; Alw: A. lanatum water extract.

ABTS radical scavenging activity

The ABTS ˙⁺ process is based on the reduction of the 2, 2 ′- azinobis (3- ethylbenzothiazoline- 6- sulfonate) radical. ABTS ˙⁺ is frequently used to define the antioxidant capacity of natural samples based on their ability to decrease the radical cation. Reactions of ABTS ˙⁺ with free radical scavengers found in the plant extract occur quickly and can be established by the subsequent reduction in the sample absorbance at 734 nm. A. lanatum samples were effective and fast scavengers of the ABTS radical, and this activity was comparable to that of ascorbic acid. The aqueous sample with an IC₅₀ of 8.0 µg/mL (95% CI: 7.4-8.6 µg/mL) showed higher activity than ascorbic acid with an IC₅₀ of 15.0 µg/mL (95% CI: 14.3-15.7 µg/mL), while the ethanol extract presented an IC₅₀ = 21. 0 µg/mL (95% CI: 20.3-21.8 µg/mL) (Table 1).

β- Carotene bleaching inhibition activity

The antioxidant activity of the tested samples, as determined by the β-carotene bleaching assay, is shown in Table 1. In the β-carotene bleaching assay, A. lanatum samples showed moderate to high antioxidant activity. The highest value was observed in the water fraction, with an IC₅₀ of 50.0 µg/mL (95% CI: 49.3-50.4 µg/mL), followed by the ethanol fraction, with an IC₅₀ of 63.0 µg/mL (95% CI: 61.2-64.7 µg/mL). The antioxidant capacity of standard ascorbic acid; IC₅₀ = 48.0 µg/mL (95% CI: 47.2-50.2 µg/mL), slightly more active than our extracts. The findings showed significant differences (p < 0. 05) in the antioxidant activity among the studied extracts.

Phytotoxic activity

Phytoxicity on germination percentages (Gp) of T. aestivum L., R. sativus L., and L. culinaris L.

The findings of the germination percentage of radish, wheat, and lentil seeds are introduced in Table 2. The effect of A. lanatum extracts on seed germination was concentration-dependent, with germination percentages decreasing with increasing extract dose. Ethanolic and water extracts exhibit potent inhibition of radish seed germination with germination rates of about 20% at 10000 μg/mL. In addition, the ethanolic and water extracts had a moderate effect on wheat seeds, with germination rates of about 80% at 10000 μg/mL. Furthermore, the ethanolic sample had an interesting effect on lentil seeds, with germination of about 40% at 10 mg/mL, while the water sample showed a moderate effect, with 80% at 10000 μg/mL. We remark that the extracts had a greater impact on radish seeds than on wheat and lentil seeds.

Table 2.

Percentage germination of radish, wheat, and lentil in the presence of A. lanatum ethanolic (Ale) and water extracts (Alw).

Ale: A. lanatum ethanolic extract; Alw: A. lanatum water extract.

Inhibition of percentage epicotyls or coleoptiles length inhibition of T. aestivum, R. sativus, and L. culinaris in the presence of A. lanatum ethanolic (Ale) and water extracts (Alw)

Regarding the wheat seeds, the ethanolic sample moderately inhibited coleoptile growth at 625 μg/mL, 1250 μg/mL, and 2500 μg/mL, while at higher concentrations, the inhibition percentages exceeded 70%. The water sample stimulated coleoptile growth by 23.6% at 625 μg/mL, 39.4% at 1250 μg/mL, and 60.78% at 2500 μg/mL; whereas at 5000 μg/mL and 10000 μg/mL, the extract inhibited growth by 20% and 85.5%, respectively (Fig. 2a). The water extract showed significant inhibition of radish stem length at all concentrations, ranging from 65.53% at 0.625 mg/mL to 97.77% at 10000 μg/mL. The ethanolic sample also exhibited significant inhibition of radish stem length, with inhibition levels lower than those of the water extract, ranging from 56.73% at 625 μg/mL to 93.73% at 10000 μg/mL (Fig. 2b). The inhibition percentage of lentil stems increased with increasing concentration of both extracts tested. The ethanolic extract showed greater inhibition than the water extract at 625 μg/mL, 1250 μg/mL, and 2500 μg/mL, whereas at 5000 μg/mL and 10000 μg/mL the water sample showed a higher inhibition percentage (Fig. 2c).

Inhibition or stimulation of root length of T. aestivum L, R. sativus L., and L. culinaris L. in the presence of (Ale) and (Alw)

For the wheat seeds, the results showed that the inhibition percentages of the ethanolic extract varied from 5.95% (at 625 μg/mL) to 84.2% (at 10000 μg/mL). However, the aqueous sample stimulates root growth with 69.25%, 87.36%, and 90% at 625 μg/mL, 1250 μg/mL, and 2500 μg/mL, respectively. At 5000 μg/mL, the stimulation of root growth decreased to 34.09%, and the highest concentration, 10000 μg/mL, inhibited the root growth with 88.97% (Fig. 3A). The extracts tested showed significant inhibition on the radish root length (more than 50% at all the tested concentrations (Fig. 3B). The water extract exhibited percentages of inhibition more than the ethanolic one; they increased from 68.53% (at 625 μg/mL) to 100% at 10000 μg/mL. In comparison, in the ethanolic extract, the percentages varied from 56.8% (at 625 μg/mL) to 87.55 % (at 10000 μg/mL). The same effect was observed for the lentil seeds. Thus, the ethanolic sample moderately inhibited root growth, with inhibition ranging from 32.04% to 68.42%. Whereas the water extract stimulated root growth at low concentrations and inhibited it at high concentrations (Fig. 3C), at 625 μg/mL mg/mL and 1250 μg/mL, root growth stimulation was 20.81% and 41.63%, respectively. At 2500 μg/mL, we observed that root length was inhibited by 10.35% and increased with increasing concentration.

Fig. 3.

Percentage root length inhibition or stimulation of wheat (A), radish (B), and lentil (C) in the presence of A. lanatum ethanolic (Ale) and A. lanatum water extracts (Alw). Negative inhibition values represent growth stimulation. Each bar represents the average ± SE with 3 repetitions (n = 30). The letters (a–h) on the bars imply significant differences (p < 0.05).

LC-HRMS analysis of A. lanatum ethanol and water extracts

The A. lanatum extracts were analyzed using LC-HRMS to detect the compounds. Mass spectrometry data were acquired using both positive and negative electrospray ionization modes. The detected mass-to-charge (m/z) values of metabolites in A. lanatum ranged from 144.0998 to 925.4789. Table 3 summarizes the chemical composition of A. lanatum ethanol and water extracts, revealing 31 and 35 potential phytochemical compounds, respectively. According to currently available literature, these metabolites appear to be reported for the first time in A. lanatum. However, compound identification was putatively assigned based on LC–HRMS data following MSI level 2 guidelines. The identified phytochemicals mainly include polyols, flavonoids, flavonoid glycosides, tannins, quinic acid, anthraquinones, carboxylic acids, phenolic compounds, triterpenoid saponins, phosphoshikimic acid, dihydroxyanthraquinones, fatty acyls, iridoids, and terpenoids, among others. The chromatograms of the extracts are shown in Fig. 4 (a, b), and the chemical structures of their major compounds are illustrated in Fig. 5.

Table 3.

Chemical composition in A. lanatum ethanol and water extracts (Ale and Alw) using LC-HRMS.

	Compounds	Class compound	RT(min)	Formula	[M-H]+ (m/z)	[M-H]- (m/z)	Relative area (%)
							Ale	Alw
1	Quinic acid	Polyol	1.421	C7H12O6		191.0545			0.78
2	Retronecine	Alkaloid	1.451	C8H13NO2	156.0999		8.89
3	Heterodendrin	Glycoside	1.453	C11H19NO6	262.1252		1.82
4	β -D-Glc-(1->4)-α-L-Rha-(1->3)- β -D-Glc	Glycoside	1.479	C18H32O15		533.1712			0.54
5	Lotaustralin	Glycoside	1.773	C11H19NO6	262.1261				0.66
6	3 beta, 6 beta-dihydroxynortropane	Alkaloid	1.817	C7H13NO2	144.0998		0.51
7	Neuraminic acid	Organic acid	2.203	C9H17NO8	268.1002		0.43
8	8-Methyldihydrochelerythrine	Alkaloid	5.396	C22H21NO4	364.1571		0.55
9	Chlorogenoquinone	Organic acid	5.557	C16H16O9		351.0739			1.95
10	Leonuriside A	Phenylpropanoid	5.631	C14H20O9	355.098		0.61
11	Feruloyl-2-hydroxyputrescine	Phenylpropanoid	5.641	C14H20N2O4	303.131				0.51
12	Isobiflorin	Glycoside	5.819	C16H18O9		353.0862			3.11
13	Apigenin 7-[rhamnosyl-(1->2)-galacturonide]	Flavonoid	6.368	C27H28O15		591.1333			0.79
14	Luteoforol	Flavonoid	6.609	C15H14O6	313.0678		0.024
15	5-O-Feruloylquinic acid	Organic acid	6.728	C17H20O9		367.1019			0.53
16	Allocryptopine	Alkaloid	6.888	C21H23NO5	392.1501		0.38
17	10-Hydroxy-8-nor-2-fenchanone glucoside	Terpenoid	6.956	C15H24O7		361.1494	2.41		1.42
18	3-(4-Hydroxyphenyl) propionic acid	Organic acid	7.368	C9H10O3		165.0541			0.55
19	3-Methylellagic acid 8-rhamnoside	Polyphenol	7.497	C21H18O12		463.0878			10.22
20	3-Oxo-12,18-ursadien-28-oic acid	Terpenoid	7.648	C30H44O3	453.3388		2.11
21	Kuwanon Z	Flavonoid	7.791	C34H26O10	593.1453				1.53
22	Myricetin 7-rhamnoside	Flavonoid	7.833	C21H20O12	463.0874		0.45		10.75
23	Luteolin 4’-O-glucoside	Flavonoid	7.891	C21H20O11		447.0941			26.56
24	Maritimetin	Flavonoid	7.897	C15H10O6	287.0523		6.57	3.55
25	4-Hydroxy-3-(2-hydroxyethyl) acetophenone 4-glucoside	Glycoside	8.009	C16H22O8		341.1232		0.80
26	6-C-Galactosylluteolin	Flavonoid	8.138	C21H20O11	449.1046		0.9	1.60
27	Bilobetin	Flavonoid	8.241	C31H20O10		551.0951		0.54
28	Eriodictyol 7-(6-galloylglucoside)	Flavonoid	8.248	C28H26O15		601.1199		0.85
29	Pedaliin	Flavonoid	8.257	C22H22O12		477.1036		3.72
30	Kaempferol 3-O-β-D-galactoside	Flavonoid	8.329	C21H20O11		447.0888	13.74
31	1,3,5,8-tetrahydroxy-6-methoxy-2-methylanthraquinone 8-o-b-d-glucoside	Quinone	8.447	C22H22O12	479.115			0.49
32	2’’,4’’-Diacetylafzelin	Flavonoid	8.468	C25H24O12		515.1192		5.47
33	5’-Butyrylphosphoinosine	Nucleoside derivative	8.611	C14H19N4O9P		477.1034		0.61
34	8-C-Galactosylluteolin	Flavonoid	8.612	C21H20O11		447.0927		5.01
35	4-O-Caffeoyl-3-O-feruloylquinic acid	Organic acid	9.165	C26H26O12		529.1341		0.75
36	Emodin 8-glucoside	Quinone	9.282	C21H20O10		431.0939	10.60
37	4’-O-methyl-(-)-epicatechin-3’-O-beta-glucuronide	Flavonoid	10.051	C23H26O12	517.1288		1.03
38	Quercetin	Flavonoid	10.206	C15H10O7	301.0341			3.09
39	Apimaysin	Flavonoid	10.238	C27H28O13		557.1286	0.60
40	Shikimate-3-phosphate	Organic acid	10.249	C7H11O8P		299.0175		0.80
41	Phytolaccoside D2	Terpenoid	11.089	C42H66O15		809.4318		1.73
42	Corchorifatty acid F	Fatty acid	11.173	C18H32O5		327.2141	0.72
43	Rhein	Quinone	11.507	C15H8O6		283.0224		1.72
44	Tiliroside	Flavonoid	11.709	C30H26O13		593.1246	1.80
45	7,8,3’,4’-Tetrahydroxyisoflavone	Flavonoid	11.771	C15H10O6		285.0391		2.31
46	Cynarasaponin H	Terpenoid	11.864	C47H74O18		925.4789		0.47
47	9-Chloro-10-hydroxy-hexadecanoic acid	Fatty acid	11.875	C16H31ClO3		365.2059	0.5
48	9,10-Dihydroxy-12,13-epoxyoctadecanoate	Fatty acid	12.125	C18H34O5		329.2295	10.80
49	Procyanidin B7	Polyphenol	12.215	C30H26O12	579.1427		0.41
50	Procyanidin B6	Polyphenol	12.58	C30H26O12		577.1330	11.49
51	Phytosphingosine	Amino alcohol	12.907	C18H39NO3	318.2972			1.09
52	Kaempferol 3-(2",6"-di-(E)-p-coumarylglucoside)	Flavonoid	13.648	C39H32O15		739.1608	0.50
53	Phytolaccoside A	Terpenoid	14.094	C36H56O10		647.3791	1.50	1.73
54	4’-Methylliquiritigenin 7-rhamnoside	Flavonoid	14.356	C22H24O8		439.1337	1.64
55	Isotheaflavin 3’-gallate	Flavonoid	14.383	C36H28O16		759.1432	2.02
56	2’’,3’’-Di-O-p-coumaroylafzelin	Polyphenol	14.866	C39H32O14		723.1665	14.27
57	2’’,6’’-Digalloyliriflophenone 3-C-glucoside	Polyphenol	14.928	C33H28O18		757.1252	0.56
58	Nigakilactone B	Terpenoid	15.578	C22H32O6	415.2086		2.16	0.6
59	23-Acetoxysoladulcidine	Alkaloid	20.666	C29H47NO4	496.3341		4.64
60	Valdiate	Terpenoid	21.112	C17H26O5		309.1726		1.32

RT: Retention time; Ale: A. lanatum ethanolic extract; Alw: A. lanatum water extract.

Fig. 4.

Chromatograms of the identified phytochemical constituent’s profiles in A. lanatum ethanol (a) and water (b) extracts.

Fig. 5.

Two-dimensional representation of the top-ranked docked phytoconstituents against 4-Hydroxyphenylpyruvate dioxygenase (HPPD) eluted from the High Resolution-Liquid Chromatography Mass Spectrometry (HR-LCMS) analysis of A. lanatum ethanol (Ale) and water (Alw) extracts.

The 31 components detected in the ethanol extract are presented in Table 3. The fragmentation pattern of the major identified compounds is provided in Supplementary Figure S1. The most abundant compound, based on relative peak was the phenolic compound “2’’,3’’-Di-O-p-coumaroylafzelin (relative abundance = 14.27%; m/z = 723.1665), as shown in Figure S1(E). This is followed by the flavonoid glycoside Kaempferol 3-O-β-D-galactoside (relative abundance = 13.74%; m/z = 447.0888) in Figure S1(A), the proanthocyanidin procyanidin B6 (relative abundance = 11.49%; m/z = 577.1330) in Figure S1(D), the hydroxy fatty acid 9,10-dihydroxy-12,13-epoxyoctadecanoate (relative abundance = 10.80%; m/z = 329.2295) in Figure S1(C), and the dihydroxyanthraquinone emodin 8-glucoside (relative abundance = 10.60%; m/z = 431.0939) in Figure S1(B).

Table 3 presented the 35 components identified in the A. lanatum water extract which the major ones are the flavonoid glycosides: luteolin 4’-O-glucoside (relative abundance = 26.56%; m/z = 447.0941) Figure S1(F), myricetin 7-rhamnoside (relative abundance = 10.75%; m/z = 463.0874) Figure S1 (G), the tannin 3-methyl ellagic acid 8-rhamnoside (relative abundance = 10.22%; m/z = 463.0878) Figure S1 (H). Furthermore, five common compounds were identified in the two extracts: 10-hydroxy-8-nor-2-fenchanone glucoside, myricetin 7-rhamnoside, maritimetin, nigakilactone B, and phytolaccoside A.

Molecular docking studies

Docking validation

To validate the molecular docking procedure, the docked native ligand structure was superimposed to the native ligand obtained from the receptor crystal structure. The resulting superposition (Fig. 6) demonstrated a minimal root mean square deviation (RMSD) between the two ligands (depicted in cyan and yellow), confirming the reliability of the docking method.

Fig. 6.

The two superimposed ligands (yellow color: co-crystallized ligand and cyan color: re-docked native ligand) in the active sites of: ‘4-Hydroxyphenylpyruvate dioxygenase (HPPD) (PDB: 6J63)’ (a) and ‘Human Peroxiredoxin 5 (PDB: 1HD2)’ (b).

Docking results analysis

Molecular docking is a powerful method, frequently employed to gain significant insight into the possible molecular mechanisms of biologically or pharmacologically active substances. To corroborate the experimental data and understand the potential mechanism underlying the allelophatic activity, molecular docking studies of the major abundant compounds were carried out using 4-Hydroxyphenylpyruvate dioxygenase (HPPD), an essential target protein for herbicide and drug development. Interestingly, the HPPD–NTBC complex structure comprised four molecules (two dimers per asymmetric unit), and the structural superposition of the chains (A, B, C, and D) within a single asymmetric unit showed no significant differences among them in one asymmetric unit²³. Thus, HPPD (PDB: 6J63) was selected as the target receptor because it is the molecular target for molecules with postemergence herbicidal activity. 4-Hydroxyphenylpyruvate dioxygenase (HPPD) was selected as a target receptor in the present study because it is an important target protein for herbicide development. This enzyme was chosen as the target receptor because it is the molecular target for compounds with postemergence herbicidal activity. Thus, in plants, the inhibition of this protein results in the depletion of carotenoids, and therefore, the absence of chloroplast growth in emerging foliar tissues results in necrosis and death^24,25. Additionally, to elucidate the mechanisms underlying the antioxidant activity of the primary metabolites detected in this work, docking simulations were performed using the human peroxiredoxin 5 enzyme (PDB: 1HD2).

Molecular docking study against HPPD (PDB: 6J63)

The results depicted in Table 4 demonstrate that among all docked ligands, the most bioactive compound is Procyanidin B6.

Table 4.

Binding energy of the docked products in the binding cavity of 4-Hydroxyphenylpyruvate dioxygenase (HPPD) (PDB: 6J63).

Compounds	Binding energy (kcal/mol)
3-Methylellagic acid 8-rhamnoside	-9.7
Myricetin 7-rhamnoside	-9.8
Luteolin 4’-O-glucoside	-9.3
Kaempferol 3-O-β-D-galactoside	-8.8
Emodin 8-glucoside	-9.1
9,10-Dihydroxy-12,13-epoxyoctadecanoate	-5.6
Procyanidin B6	-10.6
2’’,3’’-Di-O-p-coumaroylafzelin	-10.4
Sulcotrione (Standard)	-7.7
Co-crystallized ligand	-9.2

It showed the best docking score (binding energy value = -10.6 kcal/mol). The 3D diagram in Fig. 7 exhibited that this ligand fits well in the binding cavity of HPPD (PDB: 6J63). Indeed, the involved interactions, which demonstrate the formation of three hydrogen bonds between the hydroxyl groups and the His226, Gln307, and Phe424 amino acid residues, reduced the binding energy score and stabilized the Receptor-Ligand complex. Other interactions are also included, such as Pi-Cation (Phe381), Pi-Sigma (Leu368 and Leu427), Pi-Pi Stacked (Phe381), and Pi-Alkyl (Met335).

Fig. 7.

2D model of different interactions formed by the most active compound ‘Procyanidin B6’ within the active site of 4-Hydroxyphenylpyruvate dioxygenase (HPPD) (PDB: 6J63).

On the other hand, Figs. 8 and 9 show that 2’’,3’’-Di-O-p-coumaroylafzelin (−10.4 kcal/mol) was the second most active phytocompound. This docked ligand formed four H-bond interactions with Pro336, Gly366, Gln379, and Gly420 via its hydroxy groups, Pi-Sulfur with Met335, and Pi-Pi Stacked with Phe381. As shown in the tabulated results as well as in Figs. 8 and 9, it is noted that, except for "9,10-Dihydroxy-12,13-epoxyoctadecanoate," all docked compounds have higher scores than the reference (Sulcotrione) and also they showed more significant affinities than the co-crystallized redocked ligand with the exception of the 3 phytoligands: Kaempferol 3-O-β-D-galactoside (-8.8 kcal/mol), Emodin 8-glucoside (-9.1 kcal/mol) and 9,10-Dihydroxy-12,13-epoxyoctadecanoate (-5.6 kcal/mol). These significant outcomes are demonstrated and justified by the interesting interactions involved.

Fig. 8.

2D model of different interactions of: 3-Methylellagic acid 8-rhamnoside (a); Myricetin 7-rhamnoside (b); Luteolin 4’-O-glucoside (c); and Kaempferol 3-O-β-D-galactoside (d) within the active site of 4-Hydroxyphenylpyruvate dioxygenase (HPPD) (PDB: 6J63).

Fig. 9.

2D model of different interactions of Emodin 8-glucoside (e); 9, 10-Dihydroxy-12,13-epoxyoctadecanoate (f); 2’’, 3’’-Di-O-p-coumaroylafzelin (g) and (h) Sulcotrione (Standard) within the active site of 4-Hydroxyphenylpyruvate dioxygenase (HPPD) (PDB: 6J63).

Molecular docking study against human peroxiredoxin 5 (PDB: 1HD2)

Some docked phytocompounds showed more interesting docking scores than the standard reference (Ascorbic acid) and also compared to the co-crystallised ligand as shown in Table 5.

Table 5.

Binding energy of the docked products in the binding cavity of human peroxiredoxin 5 (PDB: 1HD2).

Compounds	Binding energy (kcal/mol)
3-Methylellagic acid 8-rhamnoside	-3.4
Myricetin 7-rhamnoside	-5.4
Luteolin 4’-O-glucoside	-5.1
Kaempferol 3-O-β-D-galactoside	-4.8
Emodin 8-glucoside	-5.1
9,10-Dihydroxy-12,13-epoxyoctadecanoate	-4.3
Procyanidin B6	-3.8
2’’,3’’-Di-O-p-coumaroylafzelin	-4.7
Ascorbic acid (Standard)	-3.9
Co-crystallized ligand	-4.1

For more details, it may be observed from the molecular docking outputs that Myricetin 7-rhamnoside (−5.4 kcal/mol) was identified as the most potent inhibitor of the targeted enzyme by interacting favorably with its catalytic site via four H-bonding interactions with Thr44, Gly46, Cys47, and Ser115 in addition to Pi-Donor hydrogen bonds with Thr147 and Alkyl/Pi-Alkyl contacts with Leu116 and Leu149 (Fig. 10).

Fig. 10.

2D model of different interactions formed by the most active compound ‘Myricetin 7-rhamnoside’ within the active site of Human peroxiredoxin 5 (PDB: 1HD2).

The second most effective ligand among the selected molecules ‘Luteolin 4’-O-glucoside’ (−5.1 kcal/mol) was able to build four H-bonds with Thr44, Gly46, Cys47, and Thr147 besides a Carbon hydrogen bond (Thr44), Pi-Pi T-shaped (Phe120) and Pi-Alkyl (Pro45 and Ile119), the same docking score was observed with Emodin 8-glucoside which formed Two H-bonds with Asp145 and Thr147, Carbon hydrogen bond (Thr147) and Alkyl/Pi-Alkyl interactions (Pro45 and Phe120) (Figs. 11 and 12). All the above results showed that the predominant constituents of A. lanatum extracts exhibit a strong tendency to inhibit HPPD and Human peroxiredoxin five enzymes.

Fig. 11.

2D model of different interactions of 3-Methylellagic acid 8-rhamnoside (a); Luteolin 4’-O-glucoside (b); Kaempferol 3-O-β-D-galactoside (c) and Emodin 8-glucoside (d) within the active site of Human peroxiredoxin 5 (PDB: 1HD2).

Fig. 12.

2D Model of different interactions of 9, 10-Dihydroxy-12,13-epoxyoctadecanoate (e); Procyanidin B6 (f); 2’’, 3’’-Di-O-p-coumaroylafzelin (g) and Ascorbic acid (Standard) (h) within the active site of Human peroxiredoxin 5 (PDB: 1HD2).

Predictive ADME analysis

The forecasting of ADME (absorption, distribution, metabolism and excretion) data of the selected phytocompounds have been estimated and the predicted descriptors including their pharmacokinetic and druglikeness properties are showed in Table 6.

Table 6.

In silico ADME analysis of the selected constituents : 3-Methylellagic acid 8-rhamnoside (A), Myricetin 7-rhamnoside (B),Luteolin 4’-O-glucoside (C), Kaempferol 3-O-β-D-galactoside (D), Emodin 8-glucoside (E), 9,10-Dihydroxy-12,13-epoxyoctadecanoate (F), Procyanidin B6 (G) and 2’’,3’’-Di-O-p-coumaroylafzelin (H).

Entry	A	B	C	D	E	F	G	H
GI absorption*	Low	Low	Low	Low	Low	High	Low	Low
BBB permeant *	No	No	No	No	No	No	No	No
P–gp substrate *	No	No	Yes	No	Yes	No	No	No
CYP1A2 inhibitor*	No	No	No	No	No	No	No	No
CYP2C19 inhibitor*	No	No	No	No	No	No	No	No
CYP2C9 inhibitor*	No	No	No	No	No	No	No	Yes
CYP2D6 inhibitor*	No	No	No	No	No	Yes	No	No
CYP3A4 inhibitor*	No	No	No	No	No	No	Yes	No
Log Kp (cm/s) a*	−9.03	−8.77	−8.00	−8.52	−8.29	−5.74	−8.15	−9.40
Lipinski**	Yes	No	No	No	Yes	Yes	No	No
Veber**	No	No	No	No	No	No	No	No
Egan**	No	No	No	No	No	Yes	No	No
Bioavailability Score**	0.17	0.17	0.17	0.17	0.55	0.56	0.17	0.17
TPSA (Å2)***	189.26	210.51	190.28	190.28	173.98	90.29	20.76	284.36
Consensus Log Po/w****	0.45	−0.06	0.14	−0.06	−0.07	3.29	1.46	1.25

a : skin permeability, Pharmacokinetics*, Druglikeness**, Physicochemical Properties*** and Lipophilicity****.

Some of the tested ligands were found to correctly meet the Lipinski’s rule, and so share ‘topological polar surface area’ (TPSA) values of 20.76 to 284.36 Ų, supporting the probability to have predicted high passive oral absorption, and that is expressed by the consensus Log Po/w in the range 3.29 to 4.44. In addition, the bioavailability scores ranged from 0.17 to 0.56 indicates higher bioactivity of the tested ligand. As noticed in the tabulated data, there is no P-glycoprotein (P–gp) substrate, this finding suggestes the good intestinal absorption of ligands and bioavailability. Further, all tested ligands displayed low gastrointestinal absorption (GI) except ‘9,10-Dihydroxy-12,13-epoxyoctadecanoate’. Any compound was predicted to cross the blood–brain barrier (BBB). In addition, almost all tested compounds were found to no inhibit the main cytochrome (CYP 450) enzymes; CYP1A2, CYP2C19, CYP2C9, CYP2D6 and CYP3A4. Understanding how molecules interact with cytochrome P450 (CYP) enzymes is also essential, as this family of isoenzymes is a key element in drug elimination through metabolic biotransformation. It is worth noting that between 50% and 90% of therapeutic molecules are substrates of five major isoforms (CYP1A2, CYP2C19, CYP2C9, CYP2D6, and CYP3A4). Inhibition of these isoenzymes is certainly one of the major causes of drug-related pharmacokinetic interactions that can lead to toxic or other adverse effects. The radar plot (Fig. 13), shows that all tested molecules are almost in the pink zone, confirming their better drug-likeness with a good bioavailability profile.

Fig. 13.

Bioavailability radar of the selected phytoconstituants : 3-Methylellagic acid 8-rhamnoside (A), Myricetin 7-rhamnoside (B), Luteolin 4’-O-glucoside (C), Kaempferol 3-O-β-D-galactoside (D), Emodin 8-glucoside (E), 9,10-Dihydroxy-12,13-epoxyoctadecanoate (F), Procyanidin B6 (G), 2’’,3’’-Di-O-p-coumaroylafzelin (H).

Discussion

The extraction yields for the ethanol and aqueous extracts were 2 g and 3.5 g, respectively. The initial chloroform extraction likely removed a substantial fraction of lipophilic and volatile metabolites, which is consistent with the recognized chemical diversity and richness in essential oils and non-polar constituents reported for Apiaceae species. Previous studies have documented the abundance of volatile and bioactive compounds in Apiaceae plants.

Ref.^26–28 Several new studies have indicated that many species of the Apiaceae family are excellent sources of bioactive phytochemicals with high bioavailability, such as antioxidant potency²⁹. Some literature has reported antioxidant activity in the Anisosciadium genus, particularly in our species, A. lanatum.

Our research found that the total phenolic content, flavonoid, and tannin contents in the water extract of A. lanatum were higher than in the ethanol extract. The total tannin content is being studied for the first time in our study. Only two previous studies tested these contents. Matar et al.¹⁷ reported that A. lanatum organic extracts, such as butanol and ethyl acetate, presented higher phenolic and flavonoid content than the aqueous one. El-Sayed et al.¹⁶ showed that the water -methanol extract had a higher flavonoid content than the pure water extract. The differences in results between these studies and ours may be due to the location of A. lanatum collection and/or to the different extraction methods. The antioxidant capacity, which means reducing oxidative stress and scavenging free radicals, can be tested using several other methods. The most commonly utilized are ABTS and DPPH. The ABTS assay, introduced in 1993, offers greater potential than DPPH due to the environment’s considerable polarity and the solubility of the solvents used³⁰. In the present research, three methods were tested to assess the antioxidant capacity of A. lanatum samples: DPPH radical scavenging activity, ABTS cation radical scavenging activity, and the β-carotene bleaching method. Our results demonstrated that the aqueous sample showed stronger antioxidant activity than the ethanol extract in all three assays. According to Matar et al.¹⁷, who tested the antioxidant assay of A. lanatum flowers, stems, and leaves by the ABTS method, the IC₅₀ of the water samples is closest to our results (IC₅₀ = 17.23 µg/mL). DPPH radical scavenging activity and the β-carotene bleaching method were used for the first time in our study. In the literature, a few papers mentioned the chemical composition of this species.

A preliminary and qualitative phytochemical screening of petroleum ether, dichloromethane, ethyl acetate, butanol, and aqueous fractions of flowers, leaves, and stems of A. lanatum demonstrated the presence of several contents of various constituents; such as steroids, flavonoids, phenolics, saponins, carbohydrates, tannins, alkaloids, anthraquinones, and cardiac glycosides at multiple levels¹⁷. All these compound classes are detected in our extracts. El-Sayed et al.¹⁶ tested the lycopene, β-carotene, and chlorophyll a and b contents in the four A. lanatum samples. At the same time, Mahmoud and El-Sayed¹⁸ isolated a stigmasterol and a new guaiane sesquiterpene containing a rare epoxide structural element, 10β,11β−epoxy−1α,4β,5β,7αΗ- guaiane-9-one, anisosciadone, from the mixture of methylene chloride and methanol extract of A. lanatum. These later compounds were not detected in our extracts. The difference in chemical composition may be due to differences in extraction methods and the plant’s region. Approximately more than 30% of compounds detected in A. lanatum water extract are flavonoid glycosides (relative abundance = 48.89%), such as luteolin 4’ O-glucoside, which is the primary compound (relative abundance = 26.56%). It has been shown that these compounds exhibit enjoyable antioxidant activity^31,32. Luteolin O is promising in the pharmaceutical industry and in food supplements due to its lower cytotoxicity³³. Jain et al.³⁴) proved that 3-methyl ellagic acid 8-rhamnoside presented a potent antioxidant effect (relative abundance = 11.49%). Kaempferol 3-O-β-D-galactoside is the second primary compound in the A. lanatum ethanolic extract and also offers an interesting antioxidant activity³⁵. Previous research has shown that quinic acid exhibits antioxidant activity^36,37. Apigenin and its derivatives are known for their antioxidant effects in vivo and in vitro. They repressed the oxidase enzyme, inhibit the formation of malondialdehyde (MDA), regulate the pathways of redox signaling (mitogen-activated protein kinase and phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)), enhance the neutralization of free radicals, improve the metal chelation and increase the enzymatic and non-enzymatic antioxidant, such as superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX). Apigenin and its derivatives enhance the activation of the significant regulator of oxidative stress, nuclear factor erythroid 2–2-related factor 2 (Nrf2). Apigenin reduced LPS-induced ROS production, protected mitochondrial function, and restored mitochondrial complex I activity³⁸. Propionic acid and 5-O-Feruloylquinic acid showed strong antioxidant activity^39,40. Ellagic acid and its derivatives have been shown to have antiradical activity due to their electron-donating properties. For instance, 3-O-methyl ellagic acid showed antioxidant activity (IC₅₀ = 24.28 μg/mL) against the DPPH radical³⁴. Ellagic acid, at 45 μg/mL, inhibited 71.2% lipid peroxidation of a linoleic acid, DPPH radicals (30 µg/mL; 85.6%), ABTS radicals (20 µg/mL, 93.9%), chelating capacity (45 µg/mL, 48.9 %), Hydrogen peroxide (45 µg/mL, 51.6%), and superoxide anion radicals (20 µg/mL, 65.4%)⁴¹. Myricetin, luteolin, kaempferol, apigenin, and quercetin are known for their strong antioxidant activity^32,42. Acetophenone derivatives had in vivo and in vitro antioxidant effects^43,44. Bilobetin increases antioxidant potency activating of Nrf-2 and inhibiting of Keap-1. It significantly elevates SOD activity, reduces lipid peroxidation, and decreases MDA levels⁴³.

The allelopathic effects of phytochemicals from one species on another can be harmful or beneficial affecting the germination, development, or growth of various plant parts⁴⁵. Plants release compounds from their roots, leaves, or flowers into the environment⁴⁶. The species A. lanatum was examined for its phytotoxicity against T. aestivum, R. sativus, and L. culinaris seeds. Results indicated that ethanol and water extracts significantly inhibited lentil stem growth, and radish root and stem length across all concentrations. While the water extract enhanced wheat stem and root growth at low concentrations, it showed significant inhibition at high concentrations. The observed stimulation of seedling growth at low concentrations suggests a hormetic dose–response pattern. Many experiments demonstrate that plants often exhibit biphasic responses, suggesting adaptive changes rather than experimental variability, as other studies have also shown⁴⁷. At low concentrations, compounds that are toxic to plants can induce a mild, temporary stress response in plant cells, activating defense processes. The slight increase in reactive oxygen species (ROS) acts as a trigger, leading to the activation of antioxidants and metabolic changes that promote acclimation and growth under non-lethal stress levels⁴⁸. It is noted that plants exposed to mild stress demonstrate enhanced photosynthetic performance and improved energy efficiency. This effect involves modifications in photosystem II activity and non-photochemical quenching processes. Many crop species have shown stimulated growth under low-dose stress, which can be attributed to physiological optimization⁴⁹. Overall, these findings suggest that the growth stimulation observed at low doses in this study is consistent with hormetic behavior.

The differential phytotoxic responses observed between the dicotyledones R. sativus, L. culinaris and the monocot T. aestivum may be associated with the endosperm structure and seed coat in monocots, which can regulate allelochemical entry, as well as with species-specific physiological traits. Wheat showed low-dose stimulation, consistent with hormesis; on the other hand, lentil and radish were more sensitive, attributable to greater permeability and lower detoxification potential. The phytotoxic effects of the genus Anisosciadium have not been previously studied. The allelopathic activity of A. lanatum extracts can be attributed to the varied products found in each extract. Numerous studies have reported that Apiaceae extracts possess herbicidal effects^50–54.

In this allelopathy assay, the concentrations of plant samples were selected to induce a preliminary evaluation of both stimulating and inhibitory effects under controlled conditions. In the course of these experiments, the concentrations used were within the range of those used in research to determine the potential of varying concentrations of substances and to look for positive responses to low levels of exposure. Agricultural practice often involves the application of plant extracts in a diluted form either through a foliar spray or by mixing with the soil. Due to environmental factors, the effective dose is lowered when this solution is used in the field and is reapplied⁴⁹. The findings from this study can be viewed as a trial run, providing insights for assessments and identifying areas that require further research.

The LC-HRMS analysis of A. lanatum’s water and ethanolic extracts revealed a richness in various chemical classes, including flavonoids, flavonoid glycosides, phenolic acids, terpenes, and fatty acids. The literature indicates that these secondary metabolites can inhibit the growth of certain plants^55–57. For example, quercetin inhibits root growth due to the presence of two ortho-free hydroxy groups in the C ring⁵⁷. Luteolin 7-O-β-glucuronide, at concentrations of 0.2 and 2.0 mM, reduced chlorophyll content and frond number in the species Lemna gibba⁵⁸. Myricetin and quercetin inhibited the root and shoot growth of lettuce⁵⁹. Kaempferol has been confirmed to exert allelopathic effects. Inhibition of Echinochloa colonum shoot growth increased with concentrations of 15, 30, and 60 µg/mL, yielding response indexes of 2.67, 1.81, and 0.92, respectively⁵⁹.

The phytotoxic effect is linked to either a single allelochemical or a mixture of allelochemicals present in the tested extracts. The allelopathic effect varies according to the concentration of the phytochemicals. The mechanism of action suggests that a specific component may alter cell membrane permeability and function, leading to cell death via necrosis or apoptosis⁵⁶. For instance, prior research indicated that phenolic components and a non-protein amino acid increased membrane permeability in Hyacinthus orientalis leaf tissue⁶⁰. From the molecular docking analysis, procyanidin B6 and 2’’,3’’-di-O-p-coumaroylafzelin had strong binding interactions against 4-hydroxyphenylpyruvate dioxygenase (HPPD). This implies that such phytochemicals could interfere with the catalytic activity of HPPD, a key enzyme involved in tyrosine catabolism and a general herbicidal target. The compounds’ high binding scores could be attributed to the presence of multiple hydroxyl groups and large aromatic substituents, which are favorable for hydrogen bonding, hydrophobic interactions, and π–π stacking with HPPD active-site residues. Such interactions could mimic or competitively inhibit the native substrate, thereby inhibiting enzyme activity. Myricetin 7-rhamnoside, luteolin 4’-O-glucoside, and emodin 8-glucoside exhibited strong binding affinity for human peroxiredoxin 5 (PRDX5). PRDX5 is an antioxidant protein that maintains intracellular reactive oxygen species (ROS) homeostasis and protects cells from oxidative stress. Due to their high docking scores, these anthraquinone derivatives and flavonoid glycosides may bind to the active site or regulatory sites of PRDX5, thereby stabilizing or regulating its activity. The binding affinity may be related to aromatic planar structures that increase solubility, as well as to sugar moieties that provide multiple polar contacts with amino acid side chains. These contacts may reinforce the antioxidant activity of PRDX5 or modulate its redox-sensitive signaling activities, which are of value in inflammation, neuroprotection, and cancer chemoprevention.

In summary, these results suggest a bimodal potential of the studied phytochemicals: while there are compounds that show herbicidal potential by HPPD inhibition (procyanidin B6 and di-O-p-coumaroylafzelin), there are also compounds that show affinity towards a human antioxidant enzyme that offer promise for their deployment as therapeutics or nutritional therapeutics (myricetin 7-rhamnoside, luteolin 4’-O-glucoside, and emodin 8-glucoside). These outcomes identify compound-target specificity and the structural diversity of natural compounds as a rewarding resource for agrochemical and biomedical investigation.

Conclusion

This research focused on the antioxidant activity and allelopathic effects of ethanol and water extracts of Anisosciadium lanatum on radish, wheat, and lentil seeds. The water extract demonstrated remarkable inhibition of ABTS radical cation, followed by DPPH and β–carotene bleaching. Additionally, the A. lanatum organic and aqueous extracts exhibited notable allelopathic effects on the tested crops. At low concentration, the water extract showed particular promise in stimulating root and stem wheat growth, suggesting its potential as a bio-fertilizer. This growth-promoting effect is attributed to the diverse phytochemicals identified through LC-HRMS analysis, with flavonoid glycosides being the predominant compounds. To further elucidate the possible mechanism of action, molecular docking studies of the major phytocompounds were conducted against the target proteins: the herbicide target 4-Hydroxyphenylpyruvate dioxygenase (HPPD) and the antioxidant target human peroxiredoxin 5. The binding energy indicated that Procyanidin B6 was the most active component because it fits well within the HPPD binding cavity. However, myricetin 7-rhamnoside was the most potent inhibitor of the human peroxiredoxin 5. These findings highlight the potential of A. lanatum extracts, particularly the water extract, as a natural source of antioxidants and growth stimulants for agricultural applications. Nevertheless, metabolite identification was tentatively assigned based on LC–HRMS data, and the biological activities were evaluated under controlled experimental conditions. Therefore, further studies involving compound isolation, quantitative validation using authentic standards, and field-based evaluations are necessary to confirm the reported effects.

Abbreviations

LC-HRMS

Liquid chromatography high resolution-mass spectrometry

Ale

Anisosciadium lanatum ethanol

Alw

Anisosciadium lanatum water

%

Percentage

FC

Folin–ciocalteu reagent

Na₂CO₃

Sodium carbonate solution

AlCl₃-6H₂O

Aluminum chloride

H₂SO₄

Sulphuric acid

µg GAE/mg

Microgram gallic acid equivalent per milligram

µg QE/mg

Microgram quercetin equivalent per milligram

µg CE/mg

Microgram catechin equivalent per milligram

DPPH

1,1-Diphenyl-2-picrylhydrazyl

ABTS

2,2’-azinobis (3-ethyl-benzothiazoline-6-sulfonic acid

HBL

Herbarium of biology laboratory

GP

Germination percentage

IPR

Inhibition percentage of roots

IPE

Inhibition percentage of epicotyls

IPC

Inhibition percentage of coleoptiles

RCSB

Research collaboratory for structural bioinformatics protein data bank

Pdb

Protein data bank

PDBQT

Protein data bank Q partial charge T atom type

HPPD

4-Hydroxyphenylpyruvate dioxygenase

mg/Kg

Milligrams per kilogrammes

IC₅₀

Inhibition concentration

μg/mL

Microgrammes per milliliters

h

Hour

Kcal/mol

Kilocalorie per mole

min

Minute

g

Gram

mL

Milliliters

°C

Degree celsius

nm

Nanometer

µg/mL

Microgram per milliliter

UHPLC-PDA

Ultraviolet high-performance liquid chromatography-photo diode array

SPSS

Statistical package for the social sciences

Author contributions

Malek Besbes: Conceptualization, Methodology, Formal Analysis, Data Curation, Writing–Original Draft Preparation, Writing – Review & Editing, Visualization, Project Administration, Funding Acquisition. Assia Hamdi: Methodology, Formal Analysis, Resources, Data Curation. Mabrouk Horchani: Software, Formal Analysis, Writing–Original Draft Preparation. Abeer Ayed Alshammari: Validation, Writing – Review & Editing, Funding Acquisition, Wasimah B. Al-Shammari: Funding Acquisition, Methodology, Formal Analysis. Dalal AlArdan: Validation, Writing – Review & Editing, Funding Acquisition. Saoussen Jilani: Methodology, Formal Analysis, Resources. Mansour Znati: Formal Analysis, Writing–Review & Editing, validation. Mouna Ghorbel: Data Curation, Writing–Original Draft Preparation, Writing – Review & Editing, Visualization. Hassiba Chahdoura: Methodology, Formal Analysis, Data Curation, Funding Acquisition. Ramzi Hadj Lajimi: Validation, Investigation, Resources. Jamil Kraeim: Writing–Original Draft Preparation, Visualization, Supervision. Hichem Ben Jannet: Investigation, Supervision, Funding Acquisition. All authors have read and agreed to the published version of the manuscript.

Funding

The authors declare that financial support was received for the research and publication of this publication. This research was supported by the University of Ha’il, grant number RG- 24-081.

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Declarations

Competing interest

The authors declare no competing interests.

Ethics approval and consent to participate

The plant material (Anisosciadium lanatum) belongs to the Apiaceae, which is approved for human consumption. A. lanatum is not listed as an endangered or protected species under the IUCN Red List or CITES. The extract was prepared according to the European Pharmacopoeia standards as described in the Materials and Methods section. This research did not include any experiments on vertebrate animals or humans. For experiments on Anisosciadium lanatum, no permission from an animal ethics committee is necessary.