# Phytochemical composition, phytotoxicity, and ADME modeling of Artemisia absinthium L.: implications for food safety and pharmaceutical applications

Asmae Hbika, Ayoub Farihi, Mohammed Benali, Fatima-Zahrae Ed-Darraz, Abdelhamid Bouyanzer, Mohammed Hawwal, Ramzi Mothana, Elkhadir Gharibi

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Asmae Hbika, Ayoub Farihi, Mohammed Benali, Fatima-Zahrae Ed-Darraz, Abdelhamid Bouyanzer, et al.. Phytochemical composition, phytotoxicity, and ADME modeling of Artemisia absinthium L.: implications for food safety and pharmaceutical applications. CyTA - Journal of Food, 2025, 23 (1), 10.1080/19476337.2025.2451980. hal-05109873

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# Phytochemical composition, phytotoxicity, and ADME modeling of *Artemisia absinthium* L.: implications for food safety and pharmaceutical applications

Asmae Hbika, Ayoub Farihi, Mohammed Benali, Fatima-Zahrae Ed-darraz, Abdelhamid Bouyanzer, Mohammed F. Hawwal, Ramzi A. Mothana & Elkhadir Gharibi

**To cite this article:** Asmae Hbika, Ayoub Farihi, Mohammed Benali, Fatima-Zahrae Ed-darraz, Abdelhamid Bouyanzer, Mohammed F. Hawwal, Ramzi A. Mothana & Elkhadir Gharibi (2025) Phytochemical composition, phytotoxicity, and ADME modeling of *Artemisia absinthium* L.: implications for food safety and pharmaceutical applications, CyTA - Journal of Food, 23:1, 2451980, DOI: 10.1080/19476337.2025.2451980

**To link to this article:** https://doi.org/10.1080/19476337.2025.2451980

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# Phytochemical composition, phytotoxicity, and ADME modeling of *Artemisia absinthium* L.: implications for food safety and pharmaceutical applications

Asmae Hbika<sup>a</sup>, Ayoub Farihi<sup>b</sup>, Mohammed Benali<sup>c</sup>, Fatima-Zahrae Ed-darraz<sup>a</sup>, Abdelhamid Bouyanzer<sup>a</sup>, Mohammed F. Hawwal<sup>d</sup>, Ramzi A. Mothana<sup>d</sup> and Elkhadir Gharibi<sup>a</sup>

<sup>a</sup>Laboratory of Applied and Environmental Chemistry, Faculty of Sciences, Mohammed First University, Oujda, Morocco; <sup>b</sup>Laboratory of Bioresources, Biotechnology, Ethnopharmacology, and Health, Faculty of Sciences, Mohammed First University, Oujda, Morocco; <sup>c</sup>ESCOM, TIMR (Integrated Transformations of Renewable Matter), Centre de Recherche Royallieu-CS, Université de Technologie de Compiègne, Cedex, France; <sup>d</sup>Department of Pharmacognosy, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia

## ABSTRACT

*Artemisia absinthium*, an aromatic herb distinguished by its characteristic bitter flavour, has been used in a variety of culinary applications. This study investigates its metabolites through phytochemical methods. The primary minerals in *Artemisia absinthium* stems are potassium (41%) and calcium (38.3%), forming most of its mineral content. The ethanolic extract exhibited the highest phenolic compound content, with 37.6 ± 0.09 mg GAE/g DE. The primary volatile compound identified was β-thujone, comprising over 26.6% of the volatile content. Palmitic acid is the predominant fatty acid in the hexanic extract (18.7%), while linoleic acid is the main fatty acid in the ethyl acetate extract (42.4%). Twenty phenolic compounds were identified, with 8-methoxyflavone, (+)-catechin hydrate, and chlorogenic acid as major compounds in dichloromethane, ethyl acetate, and ethanol extracts, respectively. ADME modeling indicating that 8-methoxyflavone, a major component in the dichloromethane extract, is a promising candidate for drug development targeting the central nervous system.

## ARTICLE HISTORY

Received 5 August 2024

Accepted 6 January 2025

## KEYWORDS

*Artemisia absinthium*;
phytochemical composition;
volatiles compounds; fatty
acids; phenolic compounds;
phytotoxicity; ADME
modeling

## Introduction

The plant kingdom represents an invaluable source of chemical diversity, producing countless organic compounds as well as trace quantities of inorganic compounds. These compounds are meticulously classified according to their functions and potential roles within the plant. The primary classification includes two categories: (i) primary metabolites essential for plant growth and survival and (ii) secondary metabolites, which are more specific and serve various functions. These compounds are abundant in plants, playing key roles in defense against diseases and predators. Their interactions and functions are tied to these metabolites. Plants also absorb inorganic minerals from their environment for growth and tissue development. These minerals act as structural elements in macromolecules, cofactors in enzymatic reactions, osmotic solutes for water potential, or ions for cellular charge balance (Bhatti et al., 2017). Volatile organic compounds (VOCs) play an important role in plant communication, as they are the main means of facilitating underground and above-ground interactions between plants and surrounding organisms (Effah et al., 2019). Phenolic compounds play a crucial role in protecting plants against interferers and predators (Ferrazzano et al., 2011). These compounds are gaining attention for their potential in treating and preventing diseases. Fatty acids, whether as part of larger molecules or independently, play key roles in cells, serving as structural components of membranes, energy sources, and precursors for signaling molecules (De Carvalho & Caramujo, 2018). Certain compounds pose risks

due to their phytotoxic or cytotoxic properties. Phytotoxicity refers to a substance's ability to harm plants by inhibiting growth, causing genetic mutations, disrupting development, interfering with photosynthesis, or even causing plant death. Assessments of phytotoxicity often focus on germination and root growth as indicators of plant response to biosolids stabilization (Mendes et al., 2016).

The consumption of plant-based foods and natural extracts interests researchers and the food, pharmaceutical, and cosmetic industries (Molina-Quijada et al., 2010). Aromatic and medicinal plants, rich in beneficial molecules, are highly valued for health (Gan et al., 2010) and are often used as complementary therapies alongside conventional treatments (Nwozo et al., 2023).

*Artemisia absinthium* L. (*A. absinthium*) is a medicinal perennial shrub native to Asia, the Middle East, Europe, and North Africa (Bhat et al., 2019). It is valued for its diverse pharmacological activities, including antimicrobial, insecticidal, antiviral, hypoglycemic, hepatoprotective, wound-healing, anti-inflammatory, and cardiovascular benefits (Batiha et al., 2020). Furthermore, it demonstrates a wide-ranging spectrum of antioxidant and anticancer properties (Ali et al., 2013). *A. absinthium* grows wild (Zoubi Yassine et al., 2016) and is cultivated in Morocco (Fouad et al., 2015). It is widely used to flavour green tea. The essential oil of this plant can be used as a heart stimulant to improve blood circulation (Hbika, Bouyanzer, et al., 2022). The essential oil of *A. absinthium* from Morocco has α-thujone as its primary component, recognized as the most biologically active compound in the plant

<figure><img src="image_17.png" /></figure>

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<figure><img src="image_19.png" /></figure>

(Patočka & Plucar, 2003). While α-thujone exhibits neurotoxic properties (Pelkonen et al., 2013) and stimulates the central nervous system (Bielenberg, 2007), prolonged use of the essential oil can lead to mental and toxic disorders, including symptoms like insomnia, seizures, and hallucinations (Batiha et al., 2019). Reducing or eliminating the volatile fraction of *A. absinthium*'s essential oil could help lower the concentration of thujone. This approach would minimize the neurotoxic risks associated with thujone while retaining other potentially beneficial non-volatile compounds from the plant.

This study focuses on the phytochemical investigation of *A. absinthium*, with the main objectives being the analysis of its chemical composition, particularly minerals, fatty acids, and volatile compounds. A key aspect of the research is the quantification and identification of phenolic compounds using sequential extraction with multiple solvents – hexane, dichloromethane, ethyl acetate, acetone, and ethanol – guided by Hansen parameters, a novel approach in the study of *A. absinthium*. This method offers a deeper understanding of the plant's chemical diversity. Additionally, integrating phytotoxicity analysis and ADMET evaluation provides valuable insights into its therapeutic potential, food applications, and safety, paving the way for innovative uses of *A. absinthium*.

# Materials and methods

## Plant material

The *A. absinthium* specimens used in this study were collected in October 2023 from Zkara (34.55256, -2.1955), a secluded village in the Mestferki district in the Oujda Angad prefecture in the eastern region of Morocco (Figure 1). Oujda is approximately 25 km away from the city of Oujda. The study area has a warm Mediterranean climate

with dry summers and is classified as (Cs) according to the Köppen-Geiger classification (Peel et al., 2007).

The choice of this species is based on its abundance in the eastern region and its frequent use in local traditional medicine for the treatment of various ailments. The aerial part of *A. absinthium*, freshly collected, was dried at room temperature in a dry, well-ventilated place, protected from light and sunlight. It was then finely ground into a homogeneous matrix using an electric grinder and stored at -4°C. We opted for a particle size distribution of <250 µm.

## Ash analysis by X-ray fluorescence

The mineral content in the leaves and stems of *A. absinthium* was determined using the Shimadzu EDX-7000 X-ray fluorescence spectrometer, controlled by PCEDX software. The leaves and stems of *A. absinthium* were ashed in a muffle furnace at a temperature of 600°C for 5 h to retain only the mineral compounds.

## Extraction processes

A mass of 30 g of aerial part of *A. absinthium* was successively subjected to a series of solid-liquid extractions using a Soxhlet apparatus with solvents of increasing polarity (250 ml each): hexane, dichloromethane, ethyl acetate, acetone, and ethanol. The extraction duration varies depending on the exhaustion of all organic matter contained in the matrix for each solvent used. Each obtained extract is then vacuum-evaporated using a rotary evaporator, and the residue remaining in the Soxhlet cartridge is subsequently extracted with the next solvent, and so on, as described in Figure 2.

<figure><img src="image_20.png" /><figcaption>Figure 1. *A. absinthium* plant harvesting rental from the city of Oujda Angad in Morocco's southwest oriental region (Hbika et al., 2024).</figcaption></figure>
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<figure><img src="image_22.png" /><figcaption>Figure 2. Procedure schema for extraction.</figcaption></figure>

## Analysis of volatile compounds

The HT2000H automatic headspace sampler, obtained from HTA (Brescia, Italy), was employed for the solid-phase micro-extraction (SPME) procedure, operating in the constant-type sampling mode. Prior to initial use, the SPME fiber was conditioned at 250°C for 5 min to ensure optimal performance and remove any residual contaminants. Throughout the extraction process, the syringe temperature was maintained at 80°C to ensure consistent sampling conditions and to prevent the degradation of volatile compounds. Once the SPME process was completed, the separation and identification were carried out using a Shimadzu gas chromatography system (Kyoto, Japan), equipped with a BPX25 capillary column with a stationary phase composed of 5% diphenyl dimethylpolysiloxane (30 m × 0.25 mm internal diameter, 0.25 µm film thickness), coupled with a QP2010 mass spectrometer. Pure helium (99.99%) was used as the carrier gas at a constant flow rate of 1.69 mL/min. The injection temperature, ion source, and interface were all set to 250°C. The column oven program started at 50°C. Sample ions were ionized in the electron ionization (EI) mode at 70 eV. The GC-MS system was operated in scan mode.

For sample preparation, 1 g of powdered plant material was accurately weighed and transferred into a sealed 20 ml sample vial, which was then securely closed with a silicone/PTFE septum cap. The septum was pierced by the needle of the SPME device, allowing the fiber to be exposed in the headspace directly above the solid sample. This ensured the selective adsorption of volatile compounds from the plant material while preventing contamination from non-volatile components. The retention indexes (RIs) of the volatile compounds identified in *A. absinthium* were determined according to the Kovàts retention index method (Kovàts, 1958).

## Analysis of fatty acids

The separation and identification were carried out on a Shimadzu gas chromatography system (Kyoto, Japan) equipped with a BPX25 capillary column containing 5% diphenyl dimethylpolysiloxane (30 m × 0.25 mm internal diameter, 0.25 µm film thickness), coupled with a QP2010 MS. Pure helium (99.99%) was used as the carrier gas at a constant flow rate of 1.69 mL/min. Injection, ion source, and interface temperatures were all set at 250°C. For the column oven, the temperature program used was initiated at 50°C. Sample element ionization was conducted in EI mode (70 eV). The mass range scanned was from 40 to 300 m/z. A microliter (1 µL) of each prepared extract, diluted with the appropriate solvent, was injected in splitless mode (split ratio 90:1). All samples were analyzed in triplicate. Finally, compound identification was achieved by comparing their retention times with those of authentic standards and the mass spectrometry fragmentation patterns with those found in databases or stored in the National Institute for Standard Technology-NIST-147, 198 compounds. LabSolutions software (version 2.5) was utilized for data retrieval and processing.

## Determination of total polyphenol content

Total phenols in *A. absinthium* extracts were determined by the Folin–Ciocalteau method according to Haddou et al. (2023) with some modifications. A sample of the extract (100 µL), with a concentration of 2 mg/mL, was combined with 200 µL of Folin–Ciocalteau reagent, 2 mL of distilled water, and 1 mL of Na₂CO₃ solution (15%). After a 2-h incubation at room temperature in the dark, absorbance was measured at 765 nm using a spectrophotometer. The calibration curve was constructed using gallic acid at
<figure><img src="image_23.png" /></figure>

different concentrations (0–100 µg/mL). All experiments were replicated three times to calculate the mean and standard deviation. The amount of total phenolic compounds was expressed in milligrams of gallic acid equivalents per gram of dry extract (mg GAE/g DE).

## Analysis of phenolic compounds

The identification of phenolic compounds in the extracts was carried out using a high performance liquid chromatography (HPLC) separation module (Waters e2695) coupled with a diode array detector (PDA Waters 2998) and Empower data processing software. Chromatograms were recorded between 254 and 300 nm. A C18 column (4.6 × 250 mm, 5 µm) was employed. The mobile phase, using a gradient mode, consisted of ultrapure water/acetic acid (0.5%) (solvent A) and methanol (solvent B) with the following conditions: 0 min, 80% A, 20% B; 20 min, 0% A, 100% B; 25 min, 0% A, 100% B; 35 min, 80% A, 20% B. The flow rate was 1 mL/min, and the injection volume was 20 µL. The peaks were identified based on the matching of retention times and UV spectra with the standards (Hbika et al., 2023). We used a high-purity powdered standard, dissolving it in either ethanol or ultrapure water depending on the solubility of each standard, to prepare the standard solution.

## Phytotoxicity test

The phytotoxicity of extracts from *A. absinthium* has been evaluated using the *Lepidium sativum* germination test method. This test aims to determine the inhibitory effect of the extracts on the germination and growth of *Lepidium sativum*. Growth measurements focus on the length of the roots. As a protocol, Petri dishes (d = 90 mm) containing filter papers and 10 *Lepidium sativum* seeds were placed and evenly distributed with 7 mL of the extract solution at different concentrations (5, 4, 3, 2, 1, 500, 250, 125, 67, 32 mg/mL). For each solution, three Petri dishes were used for each test. The inhibitory effect of the extracts was established by comparing the tested groups with control groups containing either distilled water or distilled water + 2% DMSO. The experimental procedure was repeated in the same manner for different concentrations. Subsequently, the Petri dishes were stored and incubated in darkness at a temperature of 25°C for 72 h. At the end of the test period for each Petri dish, the elongation of germinated radicles was measured. The inhibition rate was calculated, and the values of the median effective concentration EC50 were determined. The activity of the extract is assessed by calculating the percentage of inhibition of germ growth. It is estimated by comparing the test batch with a control batch according to the formula:

$$ \text{Inhibition percentage} = \frac{L_c - L_t}{L_c} \times 100 \quad (1) $$

where: Lc: Rootlet length of negative control, Lt: Length of treated rootlets

Effective Concentration for 50% inhibition (EC50) is expressed in mg/L. The toxicological classes were assigned according to the class limits of the EU criteria as described in Annex VI of Directive 67/548/EEC (EC, 1991). Chemicals are categorized as “harmful” (10 < EC50 ≤ 100 mg/L), “toxic” (1 < EC50 ≤ 10 mg/L), “or very toxic” (EC50 ≤ 1 mg/L) According to

EU classification criteria described in Annex VI of Directive 67/548/EEC (Aruoja et al., 2011). In addition, for practical reasons, the toxicity category “not harmful” was added and used by us for compounds with an EC50 above 100 mg/L (Hernando et al., 2005).

## ADME modeling

In recent decades, absorption, distribution, metabolism, and excretion (ADME) modeling as a tool for rational drug design has received considerable attention from pharmaceutical scientists (Wang et al., 2015). They are the key biological processes for the determination of a drug’s pharmacokinetic parameters (Migliorati et al., 2022). These tools have become essential for predicting molecule characteristics, assessing their ability to cross cell barriers, interacting with transporters and enzymes essential for absorption/excretion, and determining their metabolic stability (Ferreira & Andricopulo, 2019). They reduce the risks of negative side effects, lower the rates of drug development failures, and enhance the probability of clinical success, making them an indispensable tool in contemporary drug discovery and development (Srivastava et al., 2022). The SwissADME platform (available online: www.swissadme) was chosen to evaluate the pharmacokinetic parameters of *A. absinthium* extracts. It made it possible to examine the physicochemical attributes of the phenolic compounds contained in *A. absinthium* extracts and their therapeutic and pharmacokinetic potential.

## Results and discussion

### Extraction yields

The total extraction yields, either calculated from the previous residue (remaining after extraction with the prior solvent) or from the initial plant powder, as shown in Table 1, ranged from 1.08% to 16.7%. ETE demonstrated the highest yields at 16.7%, followed by hexane at 5.70%. Conversely, lower yields were observed for DE (1.08%). These findings indicate that extraction yields were influenced by both the duration of the extraction process and the chosen extraction solvent. The selection of solvent profoundly influences the extraction yield and composition of bioactive compounds from aromatic and medicinal plants. Each solvent exhibits distinct polarity and affinity for specific classes of compounds, thereby impacting the efficiency and selectivity of extraction.

Comprehending the properties and attributes of solvents holds paramount importance in refining extraction protocols and acquiring extracts enriched in desired phytochemicals for diverse applications in health care, pharmaceuticals, and nutraceuticals. Hexane, renowned as a non-polar solvent, finds widespread application in extracting lipophilic compounds like essential oils and fatty acids from plant matter (Jha & Sit, 2022). Its low polarity facilitates the selective extraction of hydrophobic compounds while sparing polar ones (Coscarella et al., 2023), given that non-polar compounds are integral components of aromatic plants; the extraction yield is a crucial consideration. Ethyl acetate, characterized by its moderate polarity and minimal toxicity, serves as a polar solvent extensively employed in natural product extraction processes. Its efficacy lies in its ability to efficiently extract polar compounds, including
<figure><img src="image_24.png" /><figcaption>Table 1. Extraction yields of different extracts and Hansen parameters of solvents.</figcaption></figure>

<table><thead><tr><th rowspan="2">Extract</th><th colspan="2">Total extraction yields (%)</th><th colspan="4">Hansen parameters</th></tr><tr><th>Previous residue</th><th>Plant powder</th><th>Solvents</th><th>δ<sub>D</sub></th><th>δ<sub>P</sub></th><th>δ<sub>H</sub></th></tr></thead><tbody><tr><td>HE</td><td>5.7</td><td>5.7</td><td>Hexane</td><td>14.9</td><td>0</td><td>0</td></tr><tr><td>DE</td><td>1.08</td><td>1.02</td><td>Dichloromethane</td><td>17</td><td>7.3</td><td>7.1</td></tr><tr><td>EAE</td><td>1.34</td><td>1.25</td><td>Ethyl acetate</td><td>15.8</td><td>5.3</td><td>7.2</td></tr><tr><td>ACE</td><td>1.37</td><td>1.26</td><td>Acetone</td><td>15.5</td><td>1.4</td><td>7</td></tr><tr><td>ETE</td><td>16.7</td><td>15.2</td><td>Ethanol</td><td>15.8</td><td>8.8</td><td>19.4</td></tr></tbody></table>

HE: Hexane Extract; DE: Dichloromethane Extract; EAE: Ethyl Acetate Extract; ACE: Acetone Extract; ETE: Ethanol Extract.

phenolics, flavonoids, carotenoids, anthocyanins, and tannins, from aromatic and medicinal plants (Nawaz et al., 2020). Extracts obtained through ethyl acetate extraction are notably rich in antioxidants and antimicrobial agents, rendering them well suited for utilization across pharmaceuticals, cosmetics, and the food industry (Cavalcanti et al., 2022).

Ethanol, recognized as a polar protic solvent, enjoys widespread use in extracting bioactive compounds from plant sources owing to its versatility, safety, and cost-effectiveness. This effectiveness is reflected in the notably higher extraction yields achieved with ethanol, surpassing 16%, underscoring its prominence in the extraction process. The majority of polyphenols exhibit solubility in polar solvents, whereas non-polar solvents like hexane and acetone facilitate the extraction of lipids and oils (Salim, 2017). The polarity of the solvent is crucial in augmenting the yield of the extracted compounds. Specifically, ethanol and methanol outperform water in extracting polyphenols, owing to their superior solubility and higher polarity for phenolic compounds (Ashraf et al., 2024).

Hansen's model uses three different parameters to describe the different interactions that can occur between compounds and extraction solvents: δ<sub>D</sub> for dispersion (Vander Waals forces), δ<sub>P</sub> for polarity (related to the dipole moment), and δ<sub>H</sub> for hydrogen bonding (Lefebvre et al., 2020). The total solubility parameter is the square root of the sum of each component squared.

$$ \delta s = \sqrt{\delta_D^2 + \delta_P^2 + \delta_H^2} \quad (2) $$

Figure 3 shows the positioning of the five solvents in Hansen's space. The solvents ACE, DE, and EAE, which are in the same range in the Hansen ternary, gave similar yields. The ETE solvent, which exhibits strong hydrogen bonding, gave a good yield, suggesting that solubility was promoted by hydrogen-type interactions. For HE, whose polar force is zero, the yield is greater than that of ACE, DE and EAE, while remaining lower than that of ETE. The polar force of the solvent apparently reduces the yield of total compound extraction.

## Ash analysis by X-ray fluorescence

Minerals are absorbed by plant roots from the soil solution in ionic form and through specific transport proteins. Macronutrients and micronutrients, or trace elements, are essential elements for a plant to complete its life cycle. Adequate nutrient supply has a significant impact and crucial importance on plant growth. The results obtained through X-ray fluorescence are presented in Table 2 and show that the main minerals present in the leaves and stems of *A. absinthium* are potassium, calcium, chlorine. In smaller quantities, iron, silicon, sulfur, and phosphorus are

<figure><img src="image_25.png" /><figcaption>Figure 3. Hansen ternary.</figcaption></figure>

<table><caption>Table 2. Mineral contents in *A. absinthium*.</caption><thead><tr><th rowspan="2">Element</th><th colspan="2">Element contents %</th></tr><tr><th>Leaves</th><th>Stems</th></tr></thead><tbody><tr><td>Potassium (K)</td><td>41.0</td><td>57.9</td></tr><tr><td>Calcium (Ca)</td><td>38.3</td><td>27.7</td></tr><tr><td>Chlorine (Cl)</td><td>9.99</td><td>8.54</td></tr><tr><td>Iron (Fe)</td><td>3.36</td><td>1.63</td></tr><tr><td>Silicon (Si)</td><td>3.03</td><td>0.71</td></tr><tr><td>Sulfur (S)</td><td>1.60</td><td>1.17</td></tr><tr><td>Phosphorus (P)</td><td>1.09</td><td>1.39</td></tr><tr><td>Magnesium (Mg)</td><td>0.58</td><td>0.07</td></tr><tr><td>Titanium (Ti)</td><td>0.37</td><td>0.12</td></tr><tr><td>Bromine (Br)</td><td>0.22</td><td>0.14</td></tr><tr><td>Zinc (Zn)</td><td>0.13</td><td>0.15</td></tr><tr><td>Strontium (Sr)</td><td>0.12</td><td>0.15</td></tr><tr><td>Scandium (Sc)</td><td>-</td><td>0.15</td></tr><tr><td>Copper (Cu)</td><td>0.07</td><td>0.15</td></tr><tr><td>Rubidium (Rb)</td><td>0.02</td><td>0.02</td></tr><tr><td>Zirconium (Zr)</td><td>0.02</td><td>-</td></tr><tr><td>Vanadium (V)</td><td>0.02</td><td>-</td></tr></tbody></table>

found, while magnesium, titanium, bromine, zinc, strontium, scandium, copper, rubidium, zirconium, and vanadium are present in trace amounts. Akzhigitova et al. (2018) showed the presence of eight mineral elements in *A. absinthium* collected in Kazakhstan: K, Mg, Ca, Mn, Fe, Zn, Cu, and Na. Most of these elements are fundamental for the proper functioning of plant vital processes. The classification and content of minor and trace elements in stems and leaves are different. Other than potassium, which is more concentrated in the stems, other major and minor elements are more abundant in the leaves.

The presence of potassium in plants is essential due to its ability to activate numerous enzymes, and a high concentration of K<sup>+</sup> is key to protein synthesis at the ribosome level, justifying its high content in leaves and stems
<figure><img src="image_26.png" /></figure>

with respective percentages of 41.0% and 57.9% of the total mineral content of *A. absinthium*. Calcium content is 38.3% and 27.7% in leaves and stems, respectively. The presence of this element, or more precisely Ca²⁺, plays an important role in stabilizing the lipid bilayer and coordinating with phospholipid and phosphate groups (Maathuis & Diatloff, 2013); Calcium is vital for maintaining the health and proper functioning of muscles, nerves, blood clotting processes, and cellular activities (Azmat et al., 2023). Chlorine (Cl) is an essential element for cell multiplication and plays a metabolic role in one of the phases of light action on chlorophyll. The leaves and stems of *A. absinthium* contain chlorine contents of 9.99% and 8.54%, respectively. Vanadium is present in the leaves as 0.018% of the mineral content but is absent in the stems. So far, it has only been recognized as essential for certain microorganisms. Some heavy metals are present in *A. absinthium* with low concentrations, such as titanium, zirconium, scandium, and silicon. They are often found in plants without being recognized for having a useful role (Shahid et al., 2012).

Other elements are present in the leaves and stems but in low concentrations, such as magnesium (Mg), iron (Fe), copper (Cu), and zinc (Zn), which play a role in the formation of some enzyme components. The presence of phosphorus (P) can be associated with esterification reactions involving several side groups (Maathuis & Diatloff, 2013). The presence of rubidium has a slight stimulating effect on metabolism (Tkachenko et al., 2020). Strontium is found in the mineral matter of leaves (0.12%) and stems (0.15%). Its presence in low concentration promotes plant growth, and this effect may be related to the interaction between calcium and strontium (Qiu et al., 2021). Low levels of bromine in the leaves (0.22%) and stems (0.14%) may be due to external contamination.

## Headspace analysis of volatile compounds

The analysis of volatile compounds was conducted through headspace, where the volatile compounds in *A. absinthium*

were trapped by HS-SPME and subsequently analyzed using GC-MS instrumentation. Table 3 presents the volatile compounds identified in *A. absinthium*, along with their retention times (RT), retention indexes (RI), and corresponding peak area percentage.

A total of 21 compounds were identified, representing 95.4% of the volatile components, with a higher quantity of natural compounds being associated with monoterpenes. The main identified compounds are β-thujone (26.6%), β-myrcene (14.4%), sabinene (13.7%), camphor (13.7%), and camphene (7.27%).

Zanousi et al. (2016) identified 37 volatile components using the HS-SPME method in *A. absinthium* leaves, representing 98.5% of the total compositions. The main components were β-thujone (17.2%), β-pinene (15.5%), 1,8-cineole (12.5%), sabinene (11.6%), and α-phellandrene (9.5%). Tehrani et al. (2012) found that *A. absinthium* leaves extracted by the headspace solid-phase microextraction method contained 37 components, of which β-thujone (17.2%), 1,8-cineole (12.5%), sabinene (11.6%), α-phellandrene (9.48%), and β-pinene (15.5%) were the main constituents.

Essential oils exhibit qualitative and quantitative disparities influenced by both inherent and external factors (Dhifi et al., 2016). Within the same species, subspecies, or variety of organisms, there exist discernible discrepancies in chemical composition, manifesting as variations in their overall chemical profile attributable to genetic disparities or variations in genetic expression (Polatoglu, 2013). Thujone stands out as possibly the most biologically potent constituent, notably neurotoxic (Pelkonen et al., 2013). It exerts stimulating effects on the central nervous system (Bielenberg, 2007). Nevertheless, prolonged utilization of the essential oil derived from this plant may precipitate mental and toxic disorders in humans, characterized by clinical manifestations such as insomnia, seizures, and hallucinations (Batiha et al., 2019).

By using headspace technique, thujone levels can be reduced by eliminating the volatile fraction. This approach minimizes the neurotoxic risks posed by thujone while

<table><caption>Table 3. Volatile compounds contained in *A. absinthium*.</caption><thead><tr><th>Peak number</th><th>Volatile compound</th><th>RT (min)</th><th>RI</th><th>Peak area (%)</th></tr></thead><tbody><tr><td>1</td><td>α-thujene</td><td>4.999</td><td>908</td><td>0.89</td></tr><tr><td>2</td><td>α-pinene</td><td>5.126</td><td>914</td><td>6.16</td></tr><tr><td>3</td><td>Camphene</td><td>5.379</td><td>925</td><td>7.27</td></tr><tr><td>4</td><td>Sabinene</td><td>5.765</td><td>941</td><td>13.7</td></tr><tr><td>5</td><td>β-pinene</td><td>5.837</td><td>945</td><td>0.57</td></tr><tr><td>6</td><td>β-myrcene</td><td>6.005</td><td>951</td><td>14.3</td></tr><tr><td>7</td><td>α-phellandrene</td><td>6.265</td><td>960</td><td>0.39</td></tr><tr><td>8</td><td>α-terpinene</td><td>6.462</td><td>967</td><td>1.50</td></tr><tr><td>9</td><td>β-cymene</td><td>6.596</td><td>972</td><td>3.15</td></tr><tr><td>10</td><td>D-limonene</td><td>6.667</td><td>974</td><td>1.68</td></tr><tr><td>11</td><td>Eucalyptol</td><td>6.734</td><td>976</td><td>0.85</td></tr><tr><td>12</td><td>γ-terpinene</td><td>7.157</td><td>990</td><td>2.95</td></tr><tr><td>13</td><td>β-terpineol</td><td>7.323</td><td>995</td><td>2.53</td></tr><tr><td>14</td><td>(+)-4-Carene</td><td>7.651</td><td>1005</td><td>0.54</td></tr><tr><td>15</td><td>NI</td><td>7.712</td><td>-</td><td>0.52</td></tr><tr><td>16</td><td>Linalool</td><td>7.804</td><td>1100</td><td>0.85</td></tr><tr><td>17</td><td>α-thujone</td><td>7.968</td><td>1014</td><td>1.23</td></tr><tr><td>18</td><td>β-thujone</td><td>8.152</td><td>1019</td><td>26.6</td></tr><tr><td>19</td><td>Camphor</td><td>8.639</td><td>1032</td><td>13.7</td></tr><tr><td>20</td><td>p-menth-1-en-4-ol</td><td>9.131</td><td>1044</td><td>0.52</td></tr><tr><td>21</td><td>Germacrene D</td><td>12.828</td><td>1562</td><td>0.56</td></tr><tr><td>22</td><td>NI</td><td>14.611</td><td>-</td><td>0.31</td></tr><tr><td>23</td><td>Isoledene</td><td>14.955</td><td>1587</td><td>2.17</td></tr></tbody></table>

NI: Not Identified, RI: Retention Indexes.
<figure><img src="image_27.png" /></figure>

preserving the other beneficial compounds found in the plant.

## Identification and quantification of fatty acids by GC-MS

The chemical composition of HE and EAE of *A. absinthium* chromatograms is presented in Table 4. Gas Chromatography-Mass Spectrometry (GC-MS) analysis revealed the presence of numerous fatty acids, the content of which varied depending on the solvent used for extraction. There is a predominance of palmitic acid with 18.7% in HE, and a predominance of linoleic acid in EAE with a rate of 42.4%. Other fatty acids are present in smaller quantities, such as stearic acid, butanoic acid, capric acid, methoxyace- tic acid, acetic acid, nonanedioic acid, and linolenic acid. The main fatty acids present in *A. absinthium* collected in Kazakhstan were linoleic acid (44.8%), oleic acid (30%), and palmitic acid (14.3%) (Akzhigitova et al., 2018). Fatty acids represent pivotal bioactive compounds deemed essential for human health, given the human body's incapacity to synthe- size them endogenously. Derived from various plant sources, these compounds offer a valuable means to enhance the human diet owing to their advantageous properties (Husain & Ahmad, 2020). Palmitic acid is a saturated fatty acid synthesized endogenously and found in food. Although it is often considered to have adverse effects on chronic dis- eases, palmitic acid is an essential component of cell mem- branes, secretion, and transport of lipids, playing crucial roles in protein palmitoylation and palmitoylated signaling molecules (Agostoni et al., 2016). Linoleic acid has received much attention in recent years due to its interesting biolo- gical benefits. Indeed, this molecule has effects on reducing obesity, diabetes, atherosclerosis, and inflammation. It also possesses properties that promote bone formation and growth (Yang et al., 2015).

## Quantitative phytochemical revelation

The results of the analysis of phenolic compounds are pre- sented in Table 5. The polyphenol content in *A. absinthium* extracts was determined using the gallic acid standard curve. As per the data in Table 5, ETE showed the highest total polyphenol content (37.6 ± 0.09 mg GAE/g DE) compared to the other extracts. This was followed by ACE, which demon- strated a polyphenol content of 32.2 ± 0.30 mg GAE/g DE, whereas the HE exhibited the lowest polyphenol content, with a value of 4.05 ± 0.27 mg GAE/g DE. The choice of solvent holds paramount importance in the extraction

<table><caption>Table 5. Contents of phenolic compounds in *A. absinthium* extracts.</caption><thead><tr><th>Extract</th><th>Phenolic compounds Total (mg GAE/g DE)</th></tr></thead><tbody><tr><td>HE</td><td>4.05 ± 0.27</td></tr><tr><td>DE</td><td>11.8 ± 0.57</td></tr><tr><td>EAE</td><td>22.5 ± 0.19</td></tr><tr><td>ACE</td><td>32.2 ± 0.30</td></tr><tr><td>ETE</td><td>37.6 ± 0.09</td></tr></tbody></table>

HE: Hexane Extract; DE: Dichloromethane Extract; EAE: Ethyl Acetate Extract; ACE: Acetone Extract; ETE: Ethanol Extract; GAE/g DE: Gallic Acid Equivalent per gram of Dry Extract.

process. Traditionally, alcohols like methanol and ethanol stand out as the predominant solvents utilized for bioactive compounds extraction in conventional methods. However, a spectrum of solvents, both chlorinated and non-chlorinated, including acetone, finds application in this pro- cess (Rodriguez Garcia & Raghavan, 2022).

The phenolic compound content values in our extracts are lower than those reported by Boudjelal et al. (2020) (180.3 ± 16.2 mg GAE/g DE). Conversely, certain extracts and fractions in our study showed a higher phenolic com- pound content compared to the findings of Kružinauskaitė and Raudonė (2021), where the phenolic compound content in *A. absinthium* extracts ranged from 21.2 to 24.7 mg GAE/g DE. Our experimental results of ETE closely mirror those reported by Hbika, Daoudi, et al. (2022) who documented a phenolic compound concentration of 31.5 mg GAE/g DE in AQE.

## Chemical composition of polyphenols

The chemical constituents of *A. absinthium* extracts were analysed using high-performance liquid chromatography coupled with a diode array detector (HPLC-DAD). This ana- lysis involved comparing retention times and UV spectra with established standards. The identified polyphenols are listed in Table 6. The predominant phenolic compounds discovered in the extracts include 8-methoxyflavone (32.3%) in the dichloromethane extract (ED), (+)-catechin hydrate (16.2%) in EAE, and chlorogenic acid (53.0%) in ETE. Additionally, quercetin (37%) and sinapic acid (35.7%) were present in ACE. Other compounds identified in the *A. absinthium* extracts, though in lower proportions, include ascorbic acid, 4-hydroxybenzoic acid, 3,4-dihydroxybenzoic acid, gallic acid, caffeic acid, malic acid, rutin, naringenin, vanillic acid, coumarin, *p*-coumaric acid, naringin, salicylic acid, cinnamic acid, apigenin, 6-hydroxyflavone, kaempferol, and *trans*-chalcone.

Ivanescu et al. (2010) detected ferulic acid and kaemp- ferol in the non-hydrolyzed extract, while hydrolysis allowed

<table><caption>Table 4. The fatty acids contained in *A. absinthium* extracts.</caption><thead><tr><th>Compound</th><th>RT (min)</th><th>HE</th><th>EAE</th></tr></thead><tbody><tr><td>Butanoic acid</td><td>11.792 ± 0.01</td><td>6.50 ± 0.09</td><td>nd</td></tr><tr><td>Acetic acid</td><td>12.350 ± 0.02</td><td>5.31 ± 0.07</td><td>nd</td></tr><tr><td>Capric acid (C10:0)</td><td>13.250 ± 0.01</td><td>1.71 ± 0.09</td><td>nd</td></tr><tr><td>Nonanedioic acid</td><td>18.267 ± 0.03</td><td>nd</td><td>2.66 ± 0.03</td></tr><tr><td>Myristic acid</td><td>20.392 ± 0.02</td><td>nd</td><td>4.00 ± 0.08</td></tr><tr><td>Metoxyacetic acid</td><td>22.608 ± 0.04</td><td>2.88 ± 0.09</td><td>nd</td></tr><tr><td>Palmitic acid</td><td>23.183 ± 0.01</td><td>18.7 ± 0.08</td><td>nd</td></tr><tr><td>Marganic acid</td><td>23.633 ± 0.01</td><td>2.39 ± 0.07</td><td>nd</td></tr><tr><td>Oleic Acid</td><td>24.358 ± 0.02</td><td>1.43 ± 0.09</td><td>nd</td></tr><tr><td>Linoleic Acid</td><td>24.433 ± 0.04</td><td>1.25 ± 0.08</td><td>42.4 ± 0.01</td></tr><tr><td>Linolenic Acid</td><td>24.442 ± 0.05</td><td>nd</td><td>2.23 ± 0.08</td></tr><tr><td>Stearic Acid</td><td>24.640 ± 0.01</td><td>3.03 ± 0.07</td><td>nd</td></tr></tbody></table>

RT: Retention Time, nd: not detected, HE: Hexane Extract, EAE: Ethyl Acetate Extract.
<figure><img src="image_28.png" /><figcaption>Table 6. Phenolic compounds contained in *A. absinthium* extracts.</figcaption></figure>

<table><thead><tr><th>N°</th><th>Compound</th><th>RT (min)</th><th>DE</th><th>EAE</th><th>ACE</th><th>ETE</th></tr></thead><tbody><tr><td>1</td><td>Ascorbic acid</td><td>3.208</td><td>-</td><td>-</td><td>-</td><td>0.1</td></tr><tr><td>2</td><td>4-Hydroxybenzoic acid</td><td>3.362</td><td>-</td><td>-</td><td>-</td><td>0.54</td></tr><tr><td>3</td><td>3,4-Dihydroxybenzoic acid</td><td>7.821</td><td>-</td><td>-</td><td>-</td><td>0.72</td></tr><tr><td>4</td><td>Chlorogenic acid</td><td>8.505</td><td>-</td><td>-</td><td>-</td><td>53</td></tr><tr><td>5</td><td>Gallic acid</td><td>8.990</td><td>-</td><td>-</td><td>1.69</td><td>6.74</td></tr><tr><td>6</td><td>Caffeic acid</td><td>9.741</td><td>-</td><td>-</td><td>-</td><td>0.59</td></tr><tr><td>7</td><td>(+)-Catechin hydrate</td><td>9.945</td><td>-</td><td>16.2</td><td>-</td><td>-</td></tr><tr><td>8</td><td>Vanillic acid</td><td>11.306</td><td>-</td><td>8.85</td><td>-</td><td>-</td></tr><tr><td>9</td><td>Naringenin</td><td>12.013</td><td>-</td><td>-</td><td>-</td><td>17.6</td></tr><tr><td>10</td><td>Sinapic acid</td><td>12.939</td><td>-</td><td>-</td><td>35.7</td><td>-</td></tr><tr><td>11</td><td>Naringin</td><td>13.634</td><td>-</td><td>1.17</td><td>-</td><td>2.14</td></tr><tr><td>12</td><td>Coumarin</td><td>14.728</td><td>-</td><td>-</td><td>1.08</td><td>1.4</td></tr><tr><td>13</td><td>p-coumaric acid</td><td>15.958</td><td>-</td><td>4.61</td><td>-</td><td>-</td></tr><tr><td>14</td><td>Quercetin</td><td>16.164</td><td>6.19</td><td>5.6</td><td>37</td><td>0.45</td></tr><tr><td>15</td><td>Salicylic acid</td><td>17.188</td><td>-</td><td>-</td><td>3.83</td><td>-</td></tr><tr><td>16</td><td>Apigenin</td><td>18.495</td><td>3.57</td><td>-</td><td>-</td><td>-</td></tr><tr><td>17</td><td>6-Hydroxyflavone</td><td>19.288</td><td>-</td><td>-</td><td>0.66</td><td>-</td></tr><tr><td>18</td><td>Kaempferol</td><td>20.273</td><td>0.63</td><td>-</td><td>-</td><td>-</td></tr><tr><td>19</td><td>8-Methoxyflavone</td><td>21.788</td><td>32.3</td><td>-</td><td>-</td><td>-</td></tr><tr><td>20</td><td>Trans-chalcone</td><td>23.348</td><td>-</td><td>0.96</td><td>-</td><td>-</td></tr></tbody></table>

HE: Hexane Extract; DE: Dichloromethane Extract; EAE: Ethyl Acetate Extract; ACE: Acetone Extract; ETE: Ethanol Extract.

extraction of *p*-coumaric acid, ferulic acid, fisetin, and patuletin. Moacă et al. (2019) identified chlorogenic acid as the primary compound, with quercitrin, rutin, and isoquercitrin present at lower concentrations. Minor amounts of other compounds like *p*-coumaric acid, luteolin, gentisic acid, caffeic acid, and apigenin were also detected. Some studies reported the presence of vanillic acid (Kordali et al., 2005), gallic acid, and caffeic acid (Craciunescu et al., 2012) in *A. absinthium*. Lee et al. (2013) study found a total of 20 polyphenolic compounds in *A. absinthium* leaves, including hydroxybenzoic acids, hydroxycinnamic acids, and flavonols. The dominant compound in the leaf extract was salicylic acid, followed by myricetin, caffeic acid, gallic acid, and ferulic acid.

The variability in chemical composition is influenced by both intrinsic factors related to the plant, such as soil type, climate, harvest periods, and plant maturity, and extrinsic factors related to the extraction techniques, including extraction duration and extraction temperature. From our results and findings in other studies, it can be inferred that the levels of chemical compounds fluctuate based on geographical factors like altitude, latitude, geomorphology, and topography. Furthermore, variations are noted in relation to climatic conditions such as precipitation, temperature, and humidity. The differences are also influenced by the type of extract and the polarity of solvents utilized in the extraction process (Hayat et al., 2020).

## Phytotoxicity of extracts

Although several plants are used in the pharmaceutical field, they may now contain components with a certain level of toxicity, which can have adverse effects on consumer health. Extracts of *A. absinthium* were evaluated for their phytotoxic activities on the germination and growth of *Lepidium sativum* seed rootlets at different concentrations. The toxic effect is visible in the degree of inhibition of root length growth, as shown in Figure 4 and EC₅₀ and UT values are shown in Table 7.

EH causes significant inhibition of rootlet growth in *Lepidium sativum* L., which increases with concentration, with an EC₅₀ value of 16.4 ± 0.91 mg/L. Total inhibition was

observed above a concentration of 2 mg/mL. ACE showed total inhibition at concentrations above 3 mg/mL, with an EC₅₀ value of 1000 ± 13.4 mg/L. EAE, DE, and ETE showed less remarkable EC₅₀ values than the other extracts, with values of 305.7 ± 5.22, 343.8 ± 4.21, and 583.7 ± 8.14 mg/L, respectively.

Typically, at lower concentrations of the tested extracts, the length of *Lepidium sativum* seedling strains demonstrates minimal alteration. However, with escalating concentrations, there is a proportional decrease in their length, indicating a dose-dependent effect. The quantity of dry biomass acquired in the experiments serves as an indicator of toxicity, with ACE demonstrating lesser toxicity compared to other extracts. The order of toxicity of the chemicals was HE > EAE > DE > ETE > ACE. This discrepancy may be attributed to the high percentage of fatty acids present in HE. Indeed, phytotoxicity is the culmination of various factors, one of which includes low molecular weight fatty acids, all of which have been demonstrated to exert inhibitory effects on plant growth (Mañas & De las Heras, 2018).

## ADME modeling results

The four guidelines of ADME modeling, proposed by Chris Lipinski of Pfizer, are as follows (Hajji et al., 2022): (1) Molecular weight: MW ≤500 g/mol; (2) Number of hydrogen bond donors: (HBD) ≤5 (Mainly OH and NH groups); (3) Number of hydrogen bond acceptor: (HBA) ≤5 (Essentially the O and N atoms); (4) The lipophilicity of the molecule: logP ≤ 5.

Table 8 gives the ADME modeling results of phenolic compounds contained in *A. absinthium* extracts. With the exception of the compound naringin (11), which shows an MW >500 g/mol, HBA >10, and HBD >5, all other compounds show parameter values in agreement with Lipinski's criteria.

The blood-brain barrier (BBB) regulates homeostasis of the central nervous system (CNS) by forming a tightly regulated neurovascular unit (NVU) that includes endothelial cells (ECs), pericytes and astrocytic end feet, which together maintain normal brain function (Arvanitis et al., 2020). To create effective CNS drugs, it is crucial to successfully overcome the obstacles presented by the BBB. Similarly, in the
<figure><img src="image_29.png" /></figure>

<figure><img src="image_30.png" /><figcaption>**Figure 4.** Percentage inhibition of *Lepidium sativum* seed rootlet growth by *A. absinthium* extracts.</figcaption></figure>

<table><caption>**Table 7.** EC50 values and toxicity units for extracts obtained from the phyto-toxicity test.</caption><thead><tr><td>Extracts</td><td>EC<sub>50</sub> (mg/L)</td><td>Effect</td></tr></thead><tbody><tr><td>HE</td><td>16.4 ± 0.1</td><td>harmful</td></tr><tr><td>DE</td><td>343.8 ± 4.21</td><td>not harmful</td></tr><tr><td>EAE</td><td>305.7 ± 5.22</td><td>not harmful</td></tr><tr><td>ACE</td><td>1000 ± 13.4</td><td>not harmful</td></tr><tr><td>ETE</td><td>583.7 ± 8.14</td><td>not harmful</td></tr></tbody></table>

HE: Hexane Extract; DE: Dichloromethane Extract; EAE: Ethyl Acetate Extract; ACE: Acetone Extract; ETE: Ethanol Extract.

development of treatments for neurodegenerative diseases, the ability of certain drugs to penetrate the BBB is important (Farihi et al., 2023). Out of 20 compounds, whose parameters are in agreement with the guideline values, 7 compounds (4-hydroxybenzoic acid (2), coumarin (12), *p*-coumaric acid (13), salicylic acid (15), 6-hydroxy-flavone (17), 8-methoxyflavone (19), *trans*-chalcone (20)) are BBB permeant. They are moderately lipophilic (WLogP 0.4–6.0) and have a moderate polarity (TPSA <79 Å²). The two criteria give these compounds a high probability of accessing the CNS (Daina & Zoete, 2016). TPSA is a topological polar surface calculated by summing the tabulated surface contributions of the polar fragment (Ertl et al., 2000). WLogP designates octanol/water partition coefficient (LogP<sub>o/w</sub>) (Daina et al., 2014).

The pharmacokinetic profiles of the compounds were represented as BOILED-Egg infographic (WLOGP vs. TPSA) system (Figure 5). BOILED-Egg predicts gastrointestinal absorption of GI and BBB permeation. The white zone brings together compounds with high gastrointestinal absorption, while the yellow zone brings together permeant BBB molecules. Naringin (11) was out of range. Calculation of polarity and lipophilicity revealed that eight compounds (4-hydroxybenzoic acid (2), vanillic acid (8), coumarin (12), *p*-coumaric acid (13), 6-hydroxy-flavone (17), 8-methoxyflavone (19), *trans*-chalcone (20), and salicylic acid (15)) fall in the yellow region, with red points indicating that they possess a high probability of brain penetration and are non-substrate of P-gp (PGP-). With the exception of coumarin, found in small quantities in ACE and ETE, each compound is selectively extracted by a single solvent. 8-Methoxyflavone (19) predominates in the DE, followed by vanillic acid,

the second-ranking compound in EAE (Table 6). Similarly, for 10 compounds in the white zone, with the exception of gallic acid in ACE and ETE, and quercetin in all extracts, 8 compounds were extracted selectively by a single solvent. Ethanol is the solvent that extracts the most of these compounds (6 compounds out of 10). Only naringenin (9) was predicted not to be a P-gp substrate and BBB permeant and have low passive GI absorption. The flavonoid naringin (11) and its aglycone naringenin (9) have relatively low oral bioavailability (Guo et al., 2022) and lack the activity of non-glycosylated forms (Ofer et al., 2005). Chlorogenic acid (4), a naturally occurring non-flavonoid polyphenol which can protect against toxicities induced by chemicals of different classes such as fungal/bacterial toxins, pharmaceuticals, metals, and pesticides (Rashidi et al., 2022), is found outside the white zone and has low gastrointestinal absorption. The low overall intestinal absorption of chlorogenic acid has been attributed to the presence of active efflux transport for this compound (Mortelé et al., 2021). This compound was extracted the most, and only with ethanol (Table 6). Quercetin (14), extracted by all four solvents and predominating in ACE (37%), is on the borderline of the white zone, which may be attributed to its low solubility, chemical stability, and bioavailability (Kandemir et al., 2022).

The physicochemical properties of the compounds show acceptable flexibility, with the number of rotatable bonds in all molecules being less than 9, of which three (chlorogenic acid (4), (+)-catechin hydrate (7), and naringin (11)) have the number of hydrogen bond donors: (HBD) and number of hydrogen bond acceptors (HBA) greater than 5.

For medicinal chemistry, among the 20 compounds found, only 3 compounds (naringenin (9), apigenin (16), and kaempferol (18)) do not show any lead-likeness violations (Teague et al., 1999), any structural alert of Brenk (Brenk et al., 2008) and any pan assay interference structures (PAINS alerts) (Baell & Holloway, 2010).

The order of synthetic accessibility of the different compounds found in the extract of *A. absinthium* is as follows: 4-hydroxybenzoic acid < salicylic acid < 3,4-dihydroxybenzoic acid < gallic acid < vanillic acid < *p*-coumaric acid < caffeic acid < sinapic acid < *trans*-chalcone < coumarin <
<figure><img src="image_31.png" /><figcaption>Table 8. Evaluation of the pharmacokinetic properties (ADME) of phenolic compounds identified in *A. absinthium*.</figcaption></figure>

<table><thead><tr><th rowspan="2">N°</th><th rowspan="2">Compound</th><th rowspan="2">Formula</th><th colspan="4">Physicochemical Properties</th><th colspan="3">Lipophilicity</th><th rowspan="2">Water Sol. ESOL Log S</th><th colspan="3">Pharmacok.</th><th rowspan="2">Drugliken. Bioav. Score</th><th rowspan="2">Medic. Chem. Synt. Acc.</th></tr><tr><th>MW (g/mol)</th><th>Csp3</th><th>HBA</th><th>HBD</th><th>TPSA (Å<sup>2</sup>)</th><th>XLOGP3</th><th>WLOGP</th><th>MLOGP</th><th>GI</th><th>BBB</th><th>Pgp</th></tr></thead><tbody><tr><td>1</td><td>Ascorbic acid</td><td>C<sub>6</sub>H<sub>8</sub>O<sub>6</sub></td><td>176.12</td><td>0.5</td><td>6</td><td>4</td><td>107.2</td><td>-1.64</td><td>-1.41</td><td>-2.6</td><td>0.23</td><td>H</td><td>No</td><td>No</td><td>0.56</td><td>3.47</td></tr><tr><td>2</td><td>4-Hydroxybenzoic acid</td><td>C<sub>7</sub>H<sub>6</sub>O<sub>3</sub></td><td>138.12</td><td>0</td><td>3</td><td>2</td><td>57.53</td><td>1.58</td><td>1.09</td><td>0.99</td><td>-2.07</td><td>H</td><td>Yes</td><td>No</td><td>0.85</td><td>1</td></tr><tr><td>3</td><td>3,4-Dihydroxybenzoic acid</td><td>C<sub>7</sub>H<sub>6</sub>O<sub>4</sub></td><td>154.12</td><td>0</td><td>4</td><td>3</td><td>77.76</td><td>1.15</td><td>0.8</td><td>0.4</td><td>-1.86</td><td>H</td><td>No</td><td>No</td><td>0.56</td><td>1.07</td></tr><tr><td>4</td><td>Chlorogenic acid</td><td>C<sub>16</sub>H<sub>18</sub>O<sub>9</sub></td><td>354.31</td><td>0.38</td><td>9</td><td>6</td><td>164.8</td><td>-0.42</td><td>-0.75</td><td>-1.05</td><td>-1.62</td><td>L</td><td>No</td><td>No</td><td>0.11</td><td>4.16</td></tr><tr><td>5</td><td>Gallic acid</td><td>C<sub>7</sub>H<sub>6</sub>O<sub>5</sub></td><td>170.12</td><td>0</td><td>5</td><td>4</td><td>97.99</td><td>0.7</td><td>0.5</td><td>-0.16</td><td>-1.64</td><td>H</td><td>No</td><td>No</td><td>0.56</td><td>1.22</td></tr><tr><td>6</td><td>Caffeic acid</td><td>C<sub>9</sub>H<sub>8</sub>O<sub>4</sub></td><td>180.16</td><td>0</td><td>4</td><td>3</td><td>77.76</td><td>1.15</td><td>1.09</td><td>0.7</td><td>-1.89</td><td>H</td><td>No</td><td>No</td><td>0.56</td><td>1.81</td></tr><tr><td>7</td><td>(+)-Catechin hydrate</td><td>C<sub>15</sub>H<sub>16</sub>O<sub>7</sub></td><td>308.28</td><td>0.2</td><td>7</td><td>6</td><td>119.6</td><td>-0.11</td><td>1.16</td><td>-0.56</td><td>-2.02</td><td>H</td><td>No</td><td>No</td><td>0.55</td><td>3.6</td></tr><tr><td>8</td><td>Vanillic acid</td><td>C<sub>8</sub>H<sub>8</sub>O<sub>4</sub></td><td>168.15</td><td>0.12</td><td>4</td><td>2</td><td>66.76</td><td>1.43</td><td>1.1</td><td>0.74</td><td>-2.02</td><td>H</td><td>No</td><td>No</td><td>0.85</td><td>1.42</td></tr><tr><td>9</td><td>Naringenin</td><td>C<sub>15</sub>H<sub>12</sub>O<sub>5</sub></td><td>272.25</td><td>0.13</td><td>5</td><td>3</td><td>86.99</td><td>2.52</td><td>2.19</td><td>0.71</td><td>-3.49</td><td>H</td><td>No</td><td>Yes</td><td>0.55</td><td>3.01</td></tr><tr><td>10</td><td>Sinapic acid</td><td>C<sub>11</sub>H<sub>12</sub>O<sub>5</sub></td><td>224.21</td><td>0.18</td><td>5</td><td>2</td><td>75.99</td><td>1.46</td><td>1.4</td><td>0.73</td><td>-2.16</td><td>H</td><td>No</td><td>No</td><td>0.56</td><td>2.17</td></tr><tr><td>11</td><td>Naringin</td><td>C<sub>27</sub>H<sub>32</sub>O<sub>14</sub></td><td>580.53</td><td>0.52</td><td>14</td><td>8</td><td>225.1</td><td>-0.44</td><td>-1.49</td><td>-2.77</td><td>-2.98</td><td>L</td><td>No</td><td>Yes</td><td>0.17</td><td>6.16</td></tr><tr><td>12</td><td>Coumarin</td><td>C<sub>9</sub>H<sub>6</sub>O<sub>2</sub></td><td>146.14</td><td>0</td><td>2</td><td>0</td><td>30.21</td><td>1.39</td><td>1.79</td><td>1.65</td><td>-2.29</td><td>H</td><td>Yes</td><td>No</td><td>0.55</td><td>2.74</td></tr><tr><td>13</td><td>p-coumaric acid</td><td>C<sub>9</sub>H<sub>8</sub>O<sub>3</sub></td><td>164.16</td><td>0</td><td>3</td><td>2</td><td>57.53</td><td>1.46</td><td>1.38</td><td>1.28</td><td>-2.02</td><td>H</td><td>Yes</td><td>No</td><td>0.85</td><td>1.61</td></tr><tr><td>14</td><td>Quercetin</td><td>C<sub>15</sub>H<sub>10</sub>O<sub>7</sub></td><td>302.24</td><td>0</td><td>7</td><td>5</td><td>131.4</td><td>1.54</td><td>1.99</td><td>-0.56</td><td>-3.16</td><td>H</td><td>No</td><td>No</td><td>0.55</td><td>3.23</td></tr><tr><td>15</td><td>Salicylic acid</td><td>C<sub>7</sub>H<sub>6</sub>O<sub>3</sub></td><td>138.12</td><td>0</td><td>3</td><td>2</td><td>57.53</td><td>2.26</td><td>1.09</td><td>0.99</td><td>-2.5</td><td>H</td><td>Yes</td><td>No</td><td>0.85</td><td>1</td></tr><tr><td>16</td><td>Apigenin</td><td>C<sub>15</sub>H<sub>10</sub>O<sub>5</sub></td><td>270.24</td><td>0</td><td>5</td><td>3</td><td>90.9</td><td>3.02</td><td>2.58</td><td>0.52</td><td>-3.94</td><td>H</td><td>No</td><td>No</td><td>0.55</td><td>2.96</td></tr><tr><td>17</td><td>6-Hydroxy-flavone</td><td>C<sub>15</sub>H<sub>10</sub>O<sub>3</sub></td><td>238.24</td><td>0</td><td>3</td><td>1</td><td>50.44</td><td>3.62</td><td>3.17</td><td>1.66</td><td>-4.19</td><td>H</td><td>Yes</td><td>No</td><td>0.55</td><td>2.81</td></tr><tr><td>18</td><td>Kaempferol</td><td>C<sub>15</sub>H<sub>10</sub>O<sub>6</sub></td><td>286.24</td><td>0</td><td>6</td><td>4</td><td>111.1</td><td>1.9</td><td>2.28</td><td>-0.03</td><td>-3.31</td><td>H</td><td>No</td><td>No</td><td>0.55</td><td>3.14</td></tr><tr><td>19</td><td>8-Methoxyflavone</td><td>C<sub>16</sub>H<sub>12</sub>O<sub>3</sub></td><td>252.26</td><td>0.06</td><td>3</td><td>0</td><td>39.44</td><td>3.53</td><td>3.47</td><td>1.91</td><td>-4.12</td><td>H</td><td>Yes</td><td>No</td><td>0.55</td><td>2.95</td></tr><tr><td>20</td><td>Trans-chalcone</td><td>C<sub>15</sub>H<sub>12</sub>O</td><td>208.26</td><td>0</td><td>1</td><td>0</td><td>17.07</td><td>3.08</td><td>3.47</td><td>3.44</td><td>-3.43</td><td>H</td><td>Yes</td><td>No</td><td>0.55</td><td>2.41</td></tr></tbody></table>

HA: Heavy atoms; AHA: Aromatic heavy atoms; Csp3: Fraction Csp3; RB: Rotatable bonds; HBA: H-bond acceptors; HBD: H-bond donors; TPSA (Å²): topological Polar Surface Area; LOGP: n-octanol/water partition coefficient (log Po/w); ESOL: Estimated SOLubility; GI: Gastrointestinal Absorption; BBB: Blood-Brain Barrier; Pgp: P-glycoprotein substrates; Bioav. Score: Bioavailability Score; Synt. Acc.: Synthetic Accessibility.

<figure><img src="image_32.png" /><figcaption>Figure 5. BOILED-Egg diagram (GI absorption vs. BBB permeability) of phenolic compounds identified in *A. absinthium*.</figcaption></figure>

6-hydroxy-flavone < 8-methoxyflavone < apigenin < naringenin < kaempferol < quercetin < ascorbic acid < (+)-catechin hydrate < chlorogenic acid < naringin

In terms of druglikeness, there are seven compounds (naringenin (9), sinapic acid (10), quercetin (14), apigenin (16), 6-hydroxy-flavone (17), kaempferol (18), 8-methoxyflavone (19)) which comply with the filters imposed by pharmaceutical laboratories: Pfizer (Lipinski et al., 2012), Ghose

(Ghose et al., 1999), GSK (Veber et al., 2002), Pharmacia (Egan et al., 2000), and Bayer (Muegge et al., 2001). All seven compounds have a bioavailability score of 55%.

Pharmacokinetic properties demonstrated that 11 of the compounds are not inhibitors of cytochromes P450 1A2, P450 2C19, P450 2C9, P450 2D6, and P450 3A4. Only 8-methoxyflavone (19) has the inhibitory quality of all five cytochromes combined. Cytochromes P450 are a family of metallo-
<figure><img src="image_33.png" /></figure>

<figure><img src="image_34.png" /><figcaption>Figure 6. Bioavailability radar of ascorbic acid.</figcaption></figure>

enzymes, comprising a large number of members, responsible for the oxidative metabolism of very diverse molecules of endogenous or exogenous origin.

Physicochemical properties describing oral bioavailability are predicted using SwissADME. The pink area is the suitable zone, and meets the following criteria: LIPO (Lipophilicity in terms of XLOGP3): -0.7 < XLOPGP3 < +5.0; SIZE (in terms molecular weight): 150 g/mol < MV < 500 g/mol; POLAR (Polarity in terms of topological polar surface area): 20 Å² < TPSA < 130 Å²; INSOLU (insolubility in water in terms of log S scale): -6 < Log S (ESOL) < 0; INSATU (Unsaturation as per fraction of carbons in the sp3 hybridization): 0.25 < Fraction Csp3 < 1; FLEX (Flexibility as per rotatable bonds): 0 < Num. rotatable bonds < 9. Ascorbic acid (1) is the only compound that meets the criteria for oral bioavailability (Figure 6).

## Conclusion

This study provides a comprehensive analysis of the chemical composition and bioactive properties of *A. absinthium* extracts, highlighting their potential applications in various fields. The serial extraction using hexane, dichloromethane, ethyl acetate, acetone, and ethanol produced distinct extracts (HE, DE, EAE, ACE, and ETE), each exhibiting unique properties based on the solvent used. Furthermore, headspace analysis of the volatile compounds highlighted β-thujone (26.6%), β-myrcene (14.4%), and sabinene (13.7%) as the dominant constituents, contributing significantly to the plant's overall chemical profile and potential bioactivity.

The phytochemical analysis revealed a diverse range of major phenolic compounds, including 8-methoxyflavone (32.3%) in DE, (+)-catechin hydrate (16.2%) in EAE, and chlorogenic acid (53.0%) in ETE. Notably, quercetin (37%) and sinapic acid (35.7%) were predominant in ACE. In the non-polar fractions, palmitic acid (18.7%) was identified as the main fatty acid in HE, while linoleic acid (42.4%) dominated in EAE. Phytotoxicity assessments indicated that the extracts generally exhibited low toxicity, with the exception of the hexane extract, which showed harmful effects likely due to its high fatty acid content. ADME analyses revealed that 19 out of 20 compounds met Lipinski's criteria, with naringin being an exception due to its molecular weight (>500 g/mol) and hydrogen bond acceptor and donor counts (>10 and >5, respectively). BOILED-Egg infographic analysis (WLOGP vs. TPSA) indicated that eight compounds (4-hydroxybenzoic acid, vanillic acid, coumarin, p-coumaric acid, 6-hydroxy-flavone, 8-methoxyflavone, trans-chalcone,

and salicylic acid) are capable of permeating the blood-brain barrier. Based on our study, we can conclude that the volatile fraction of *A. absinthium*, which is rich in thujone, a compound already recognized for its antimicrobial properties, can be used as a potential natural biopesticide for organic farming, thereby promoting sustainable agricultural practices. Furthermore, by utilizing the headspace technique, thujone levels can be reduced or eliminated, addressing the toxicity associated with *A. absinthium* and making its use safer for various applications. Moreover, EAE, EAC, and EET exhibit promise as natural food preservatives, being rich in polyphenols and non-toxic, offering a safer alternative to synthetic additives. Also ED, containing 32.3% 8-methoxyflavone as the major compound, stands out for its ability to cross the blood-brain barrier. Due to its moderate lipophilicity and polarity, 8-methoxyflavone emerges as a strong candidate for the development of drugs targeting the central nervous system (CNS). Therefore, ED could be incorporated as a functional additive in beverages, providing not only flavor enhancement but also pharmaceutical benefits, such as antimicrobial effects and digestive support, with no associated phytotoxicity.

## Acknowledgments

The authors extend their appreciation to Researchers Supporting [Project number (RSP2025R119)], King Saud University, Riyadh, Saudi Arabia, for funding this work.

## Disclosure statement

No potential conflict of interest was reported by the author(s).

## Funding

The research was funded by Researchers Supporting Project number (RSP2025R119), King Saud University, Riyadh, Saudi Arabia.

## Author contributions

Conceptualization, A.H. and E.G.; methodology, A.H., A.B. and E.G.; software, A.H. and A.F.; validation, A.H., A.B. and E.G.; formal analysis, A.H. and A.F.; investigation, F.E. and M.B.; writing – original draft preparation, A.H. and E.G.; writing – review and editing, A.H., M.B., R.M., M.H and E.G.; supervision, A.B. and E.G.; project administration, R.M. M.H; funding acquisition, R.M and M.H.

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