GC-MS Analysis and Molecular Docking Studies of Lavandula dentata Leaves Extract of Taif Region, Saudi Arabia

Lavandula spp. is one of the most cultivated plants worldwide due to its essential oil properties. Several studies have demonstrated its antimicrobial and biological activities (Carrasco et al., 2016; de Rapper et al., 2016). This genus consists of 39 species, along with approximately 400 registered cultivars and several hybrids (Benabdelkader et al., 2011). The major compounds of Lavandula and its essential oil are linalool, linalyl acetate and alpha-terpineol (Speranza et al., 2023). However, the chemical composition of essential oils from the same plant species varies qualitatively and quantitatively depending on environmental conditions and their processing method (Asekun et al., 2007; Singh et al., 2008). Lavandula dentata L., belonging to the family Lamiaceae, is a widespread wild herb that grows in Saudi Arabia as well as in many other countries worldwide (Al-Sarar et al., 2014).
       
Lavandula dentata L. is a medicinal plant commonly used in traditional medicine to treat different diseases (Bouyahya et al., 2023). It was reported that L. dentata extracts and essential oils can be used as an antioxidant, anticancer, antiparasitic, antidiarrheal, antispasmodic, antihypertensive, antibacterial, antilithiasic, antifungal, anti-inflammatory and in treatment of neurological, gastrointestinal, urinary, genital, musculoskeletal and dermatological diseases (Belhaj et al., 2021; Djaballah and Bellaka, 2020; Fouad and Lahcen, 2020; Imen et al., 2021; Justus et al., 2019a, 2019b; Mohammed et al., 2020; Mrabti et al., 2019). These biological activities of L. dentata could be assigned to their bioactive compounds of flavonoids, tannins, terpenoids and anthracene derivatives (Imen et al., 2021).
       
Spectrometric and chromatographic methods to screen medicinal plants provide fundamental information on their chemical and pharmacological activities. This information is crucial in identifying plants with biological activity (Juszczak et al., 2019). Recently, gas chromatography-mass spectrometry (GC-MS) and Fourier-transform infrared (FTIR) have commonly been used for functional groups detection and identification of different bioactive therapeutic compounds that are present in medicinal plants (Fan et al., 2018; Satapute et al., 2019). However, GC-MS analysis represents one of the best, fastest and most accurate techniques for various compounds detection such as alkaloids, nitro compounds, alcohols, long-chain hydrocarbons, steroids, esters, organic acids and amino acids (Razack et al., 2015).
       
Monoamine oxidases (MAOs) are mitochondrial enzymes with two isotypes A and B. Their function is the breakdown of monoamine neurotransmitters including dopamine, adrenaline, serotonin, noradrenaline, β-phenylethylamine, norepinephrine and tyramine. MAO-A has a greater affinity for serotonin and noradrenaline as hydroxylated amines. MAOs overexpression can lead to mitochondrial damage that in turn could lead to a neurotoxic nature, depression, Alzheimer’s disease and Parkinson’s disease. Blocking MAOs with small molecules has advanced treatment for neuropsychiatric disorders and neurodegenerative diseases. Many MAO inhibitors have been developed since the 1960s, however, MAO inhibitors usage is linked to health improvement, mental/neurological diseases, off-target effects, safety concerns, dietary restrictions and low tyramine intake (Aljanabi et al., 2021; Kumar et al., 2024; Özdemir et al., 2021).
       
In the present study, we used the GC-MS technique to detect and identify the phytochemical compounds present in lavender leaves methanolic extract that is cultivated in the Taif region of Saudi Arabia. In Silico computational analysis was done using SwissADME and ProTox-II to predict the physicochemical, biological activities and predicted toxicity (lethal dose, LD₅₀) of four select components from the extract of Lavadula methanolic extract. In addition, molecular docking was done using linalool and 7-Methoxy-2-oxo-2H-chromene-3-carboxylic acid to Monoamine Oxidase A enzyme.

The experiment was conducted at Taif University in the summer season of 2023. Lavandula dentata L. were freshly collected from Taif region, Saudi Arabia (21°16'30.34"N 40°24'22.16"E). Before use, leaves were rinsed in water, dried away from the sun, collected and ground in an electric grinder to obtain fine powder. The powder was mixed with methanol (200 mL), macerated overnight and then filtered. The marc was macerated in methanol for 1 h and then filtered, this step was repeated twice. Finally, we obtained 600 ml of the collected methanol that was evaporated under vacuum at 40°C to yield 1.289 g of lavender leaves methanolic extract (LE) (Labarbe et al., 1999).
       
Gas chromatography-mass spectroscopy (GC-MS) is an ideal technique for phytoconstituents characterization to predict their structure, name, formula and retention time. RTX-5 MS capillary column (Restek) was used to chemically characterize the 95% methanolic LE with the help of GC-MS-QP2010 Plus system (Shimadzu, Japan) (Siddiqui et al., 2015). GC separates the various constituents into their components with their variable retention times and a mass spectrophotometer identifies the components. A chromatogram of relative abundance against retention time was produced by the software, coupled to the mass spectrophotometer.
       
We have selected the most abundant compounds from the extract to be used further in molecular docking analysis. The 3D conformations of selected compounds (ligands) were downloaded from the PubChem database. The 3D crystal structure of human Amine Oxidase A (receptor protein) with Harmine (actual co-crystallized ligand) (PDB ID: 2z5x with resolution 2.20 Å) was retrieved from RCSB PDB (http://www.rcsb.org/pdb/home/home.do). All water molecules, cofactor (FAD) and ligand (Harmine) were removed from the receptor protein by using PyMOL software.
       
The free online service SwissADME was used to predict their physicochemical properties, pharmacokinetics, drug-likeness and medicinal chemistry (http://www.swissadme.ch/) (Daina et al., 2014; 2017; Daina and Zoete, 2016). The best-docked compounds were selected according to Lipinski’s rule of five. Compounds that obey the Lipinski rule are considered ideal drug candidates. Lipinski stated that a compound could display drug-like behaviour if it does not fail more than one of the criteria: molecular weight not more than 500; hydrogen bond donors ≤5 and acceptors ≤10; lipophilicity less than 5 and molar refractivity between 40 and 130 (Lipinski et al., 2012).
       
Predicted toxicities and the Lethal Dose (LD₅₀) predictions of the studied compounds were obtained using a ProTox-II web server (https://tox-new.charite.de/protox_II/) (Banerjee et al., 2018). SMINA/Anaconda was used in the molecular docking of selected compounds into the binding pockets of the receptor protein. The re-docking procedure was also applied to validate the accuracy of the docking protocol (Boström et al., 2003). Docking validation was done by redocking the co-crystallized ligand (Harmine) to the active site by using the ad4_scoring function. The best-docked conformations have been chosen by comparing the root mean squared deviation (RMSD) values with the actual co-crystallized ligand against the protein (Hevener et al., 2009). After docking, all the complex images were analyzed by using the Protein Ligand Interaction Profiler (PLIP, https://plip-tool.biotec.tu-dresden.de/plip-web/plip/index) to identify non-covalent interactions between protein and their ligands and their 2D images (Adasme et al., 2021).

Gas Chromatography-Mass Spectrometry (GC-MS) analysis has become a pivotal technique in the identification and characterization of bioactive compounds in various botanical extracts (Adams, 2007). In the current study, Lavandula dentata leaves methanolic extract underwent GC-MS analysis to elucidate its chemical composition.
       
The GC-MS analysis revealed a complex mixture of more than one hundred compounds with different retention time within the methanolic extract of Lavandula dentata leaves as displayed in Fig 1 and Table 1-3. One of the major constituents identified was linalool, which is consistent with previous studies on Lavandula species (Bilia et al., 2014). Linalool has been extensively studied for its pharmacological properties, including its antimicrobial (Faleiro et al., 2003) and anxiolytic effects (Linck et al., 2009).
 

 

 

 

       
Moreover, the presence of Chromen-2-one was detected in the Lavandula dentata extract. Chromenes, also known as chromanones or chromones, represent an important class of heterocyclic compounds with diverse biological activities (Zhang et al., 2016). These compounds are characterized by their structural motif comprising a benzene ring fused to a cyclic ether ring. Chromenes exhibit a wide range of pharmacological properties, making them valuable targets for drug discovery and development. For instance, several studies have highlighted the anticancer potential of chromenes due to their ability to inhibit various cancer cell proliferation pathways (Zhou et al., 2019).
       
Additionally, chromenes have been reported to possess antioxidant properties, which can play a crucial role in combating oxidative stress-related diseases such as cardiovascular disorders and neurodegenerative conditions (Zhang et al., 2016). Furthermore, chromenes have attracted significant attention in the field of medicinal chemistry owing to their diverse biological activities. Research efforts have focused on synthesizing novel chromene derivatives with enhanced pharmacological profiles (Xu et al., 2020). These derivatives often undergo structural modifications to optimize their drug-like properties, such as improving solubility, bioavailability and target specificity.
       
Additionally, retinal was identified in the Lavandula dentata leaves extract. Retinal, a derivative of vitamin A, plays a crucial role in vision by serving as the light-absorbing chromophore in visual pigments (Palczewski, 2006). It is a vital component of the visual cycle, where it undergoes photoisomerization upon absorbing light to initiate signal transduction in photoreceptor cells. In addition to its role in vision, retinal also participates in non-visual processes, including the regulation of circadian rhythms (Buhr et al., 2015). Melanopsin-containing retinal ganglion cells in the retina utilize retinal as a chromophore to detect light and transmit signals to the brain’s suprachiasmatic nucleus, thereby synchronizing the body’s internal clock with the day-night cycle. Furthermore, retinal is essential for maintaining the health and integrity of retinal tissue. Deficiencies in retinal metabolism or transport can lead to retinal degenerative diseases such as retinitis pigmentosa and age-related macular degeneration (Sparrow et al., 2010). Understanding the biochemical pathways involving retinal metabolism is crucial for developing therapeutic interventions to treat these blinding disorders.
       
Furthermore, the GC-MS analysis detected the presence of Cumarin-3-carboxylic acid, 7-methoxy. Cumarin-3-carboxylic acid, 7-methoxy, also known as methoxycoumarin or 7-methoxycoumarin, is a derivative of coumarin with a methoxy substituent at the 7-position of the aromatic ring. This compound belongs to the broader class of coumarin derivatives and has garnered attention for its potential pharmacological activities and diverse applications in medicinal chemistry. Studies have indicated that Cumarin-3-carboxylic acid, 7-methoxy, possesses notable biological properties, including antioxidant and anti-inflammatory effects. These properties make it a promising candidate for the development of therapeutic agents targeting oxidative stress-related diseases and inflammatory conditions (Borges et al., 2016). The presence of the methoxy group at the 7-position may contribute to its antioxidant activity by enhancing its ability to scavenge free radicals and mitigate oxidative damage.
 
Moreover, Cumarin-3-carboxylic acid, 7-methoxy has been investigated for its potential anticancer properties. Research suggests that certain methoxycoumarin derivatives exhibit cytotoxic effects against cancer cells and may hold promise as anticancer agents through mechanisms such as induction of apoptosis and inhibition of proliferation (Yang et al., 2015). The structural modification of coumarin to incorporate a methoxy group at the 7-position could influence its interactions with cellular targets involved in cancer progression, thereby enhancing its therapeutic efficacy. Furthermore, methoxycoumarin derivatives have been explored for their antimicrobial activities. Studies have demonstrated the antibacterial and antifungal properties of certain methoxycoumarin derivatives, indicating their potential as antimicrobial agents for combating infectious diseases (Lee et al., 2019). The presence of the methoxy group may contribute to the compound’s ability to disrupt microbial cell membranes or interfere with essential metabolic processes, thereby exerting antimicrobial effects.
       
Four phytocompounds (linalool, retinal, chromene-2-one and 7-Methoxy-2-oxo-2H-chromene-3-carboxylic acid) were analyzed for Amine Oxidase A docking studies. Their physicochemical properties, pharmacokinetics, drug-likeness and medicinal chemistry are found in Tables 4-7. Retinal was excluded according to missing calculated parameters. Linalool, chromene-2-one and 7-Methoxy-2-oxo-2H-chromene-3-carboxylic acid met Lipinski’s conditions, which is considered to predict optimal drug-like character. They reported no Lipinski’s violations that suggested these are orally bioavailable drugs. It is reported that selected compounds have low lipophilicity, good gastrointestinal absorption and blood-brain barrier permeability (except for 7-Methoxy-2-oxo-2H-chromene-3-carboxylic acid) and bioavailability (Daina et al., 2014). 
 

 

 

 

       
In ProTox-II, linalool and 7-Methoxy-2-oxo-2H-chromene-3-carboxylic acid are assumed to be non-toxic with their predicted toxicity class and LD₅₀ 2200mg/kg (Class: 5) and 200 mg/kg (Class: 3), respectively. Linalool is assumed to be safer than 7-Methoxy-2-oxo-2H-chromene-3-carboxylic acid because it is inactive against hepato-, neuro-, nephro-, cardio-, cyto-, immuno-, respiratory toxicity, carcino- and mutagenicity.
       
For molecular docking: Linalool and 7-Methoxy-2-oxo-2H-chromene-3-carboxylic acid were docked, separately, with the receptor Amine Oxidase A. The docking scores of linalool and 7-Methoxy-2-oxo-2H-chromene-3-carboxylic acid are -24.4 and -33.7, respectively, with the receptor protein. Both are docked with nearly identical co-crystallized ligand Harmine binding site to receptor protein. However, linalool appears to be more stable in the docking site due to the presence of nine hydrophobic interactions with the receptor protein in comparison with only two hydrophobic interactions for 7-Methoxy-2-oxo-2H-chromene-3-carboxylic acid (Table 8 and 9, Fig 2). The in silico and docking computational results suggested that linalool could be a promise MAO-A inhibitor.
 

 

 

       
Computational analysis plays a critical role in the identification and development of new drugs with better therapeutic effects. Cheminformatics and molecular modeling have long been used to discover and develop better drug therapies (Noorjahan and Saranya, 2018). As part of standard drug discovery processes, in silico modeling is now an integral component. These methods aid in discovering new medications or improving the therapeutic efficacy of a chemical series in early drug development (Kumar et al., 2024; Stanzione et al., 2021). Linalool’s potential activity in the central nervous system has been studied for its mechanism of antidepressant action through monoaminergic pathways. New studies have expanded knowledge about its interaction with different targets and pathways, making it a promising natural compound for treating depression (Dos Santos et al., 2022). According to the present results, linalool could have a crucial role in the treatment of different neurodegenerative disorders through its highly effective MAO-A inhibition. Therefore, further in vitro and in vivo studies are needed to verify our assumption and report its mechanism pathway.

In conclusion, the GC-MS analysis of the methanolic extract of Lavandula dentata leaves revealed the presence of various bioactive compounds including linalool, retinal, chromene-2-one and Cumarin-3-carboxylic acid, 7-methoxy. These compounds have been associated with diverse pharmacological properties, suggesting the therapeutic potential of Lavandula dentata extract in medicine and pharmaceutical applications. Linalool has an effective inhibitory action on MAO-A according to the present in silico and docking analysis that could be a promising safe therapeutic drug for various neurodegenerative diseases. More studies are thus recommended to shed more light on the pharmacological and medical properties of Lavandula dentata.

The authors extend their appreciation to Taif University, Saudi Arabia, for supporting this work through project number (TU-DSPP-2024-283).
 
Author contributions
 
The authors contributed to the practical part, writing the original draft, editing and accepting the final version of this article.
 
Funding
 
This research was funded by Taif University, Taif, Saudi Arabia (TU-DSPP-2024-283).

The authors declare no conflict of interest.