Introduction
Plantago lanceolata L., commonly known as Ribwort plantain, is a widespread species within the Plantago genus of the Plantaginaceae family [1]. Although primarily a perennial, it can sometimes be classified as annual or biennial. This plant thrives in temperate regions, commonly growing in meadows, pastures, green spaces, and along roadsides [2]. Traditionally, various parts of P. lanceolata have been used ethnomedicinally; for example, the whole plant has been used to prepare eye lotions [3]. The aerial parts of the plant have wound-healing, anti-asthmatic, antibacterial, and anti-inflammatory properties [4]. Its leaves serve as an expectorant and are used to alleviate abdominal pain and treat inflamed wounds [5, 6]. The seeds of P. lanceolata were traditionally used in treating parasitic worms, and the plant’s mucilage, as a laxative, reduces membrane irritation [3]. In addition to its medicinal properties, P. lanceolata is utilized in various industries as a food additive, in cosmetic formulations, as an ingredient in insecticides, and for environmental applications, such as the removal of heavy metals from contaminated sites. The therapeutic potential of this species is largely attributed to the rich secondary metabolite content of its leaves, roots, and bark [7]. These valuable secondary metabolites include acteoside, flavonoids, phenylpropanoid glycosides, phenylcarboxylic acids, silica, mucilage, tannins, iridoid glycosides, zinc, and potassium salts. Among the iridoid glycosides, aucubin and catalpol are particularly prominent [8]. These compounds are recognized as chemotaxonomic markers, extensively studied across multiple plant organs [9]. Previous studies have evaluated the biocompatibility and toxicity of extracts derived from different parts of P. lanceolata [10, 11]. Further research has focused on the biological activities of P. lanceolata root extracts [12]. The study of medicinal plants’ chemical properties and biological potential is particularly crucial. The therapeutic effects of P. lanceolata are closely linked to its bioactive compounds [1]. The cost-effectiveness of petroleum ether compared to other organic solvents makes it an appealing choice as a non-polar extraction solvent [13]. It is widely utilized to extract non-polar substances such as vegetable and essential oils. P. lanceolata is known for its non-aromatic nature and low yield of essential oils. Previously, the aerial parts of this plant were subjected to hydrodistillation using a Clevenger apparatus, with the essential oils collected in n-pentane for further analysis [14]. Petroleum ether was identified as an effective solvent for extracting non-volatile lipophilic compounds from roots. On the other hand, aqueous extraction, using water as the solvent, is commonly applied across medicine, chemistry, and biology to isolate various compounds. This study aimed to identify the bioactive compounds and assess the antibacterial efficacy of P. lanceolata root extracts. Specifically, it aimed to evaluate the antibacterial properties of petroleum ether and aqueous extracts after fractionation.

Materials and Methods
Plant material 
P. lanceolata was collected from the collection site (36°41’15.5”N, 48°24’02.2”E) at the University of Zanjan, Iran, and was verified by the Department of Botany, University of Zanjan. The voucher specimen number 14253 was recorded for P. lanceolata. The root organ of the plant was cut and shade-dried at room temperature for 7-10 days. 

Extraction
A total of 250 g of powdered P. lanceolata roots underwent sequential extraction using petroleum ether and methanol via a reflux apparatus, with each solvent applied for 16 h. The methanol extract was further partitioned through liquid-liquid extraction with dichloromethane, ethyl acetate, n-butanol, and water [15]. The aqueous phase was filtered through Whatman No. 1 filter paper to remove fibrous plant debris (Figure 1).

Only petroleum ether and aqueous extracts were selected for this study, as previously reported [12] had analyzed other fractions. The final extracts were concentrated under reduced pressure using a rotary evaporator and air-dried at ambient temperature for one week.

Microorganisms culture 
Gram-positive Bacillus cereus (ATCC 11778) and gram-negative Salmonella paratyphi (ATCC 5702), and Proteus vulgaris (PTCC 1182) strains were obtained from the Department of Biotechnology, School of Pharmacy, Zanjan University of Medical Sciences, Zanjan, Iran. These bacterial cultures were grown in Mueller-Hinton broth and incubated at 37 °C for 18 h before experimentation.

Antibacterial activity assay
The disc diffusion assay was used to estimate the antibacterial potential of P. lanceolata roots, according to the National Committee for Clinical Laboratory Standards [16]. To obtain a 100 mg/mL concentration, petroleum ether and aqueous extracts were dissolved in dimethyl sulfoxide (DMSO), and the discs were impregnated with 5 µL of each extract. Gentamicin at a 10 µg/mL concentration was used as the positive control, and DMSO was used as the negative control. In this regard, the turbidity of inocula was in accordance with 1.5×108 CFU/mL, 0.5 McFarland standard [17]. The diameter of the inhibition zone (mm) was measured to estimate the antibacterial properties of the extracts. The Clinical and Laboratory Standards Institute (CLSI) guidelines were followed to determine the minimum inhibitory concentrations (MICs) by standard broth microdilution [18]. The root extracts were used at concentrations ranging from 1 to 4 mg/mL. MICs were determined as the lowest extract dose that did not exhibit visible growth in the well. The minimum bactericidal concentrations (MBCs) were evaluated by culturing 100 μL from each well on Mueller-Hinton agar plates and incubating at 37 °C for 24 h. 

Gas chromatography-mass spectrometry (GC-MS) analysis 
The petroleum ether and aqueous extracts of P. lanceolata root were analyzed by GC-MS. The conditions and temperature program are presented in Table 1, according to a previously described method [15].


Working solutions (5 mg/mL) were prepared by dissolving the dry extracts in HPLC-grade methanol. They were filtered using a sterile 0.22 μm filter before injection into the device.

Statistical analysis 
All the experiments were performed in three replicates. The data were reported as Mean±SD. The Figures were designed using Excel software, version 2016. 

Results 
Antibacterial activity 
The petroleum ether and aqueous extracts of P. lanceolata roots were tested individually against various bacterial strains. According to Table 2, these root extracts exhibited antibacterial activity against both gram-positive and gram-negative bacteria.


The petroleum ether extract demonstrated the highest antimicrobial effect, producing a 15.50 mm inhibition zone against P. vulgaris using the disc diffusion method. However, none of the extracts matched the antibacterial efficacy of the standard antibiotic gentamicin against the tested bacteria (Table 2). The petroleum ether root extract had the lowest MIC of 2 mg/mL against P. vulgaris and B. cereus. These results were further supported by MBC values, which were confirmed at 3 mg/mL (Table 2). In contrast, the aqueous extract exhibited higher MICs against the tested bacteria and was less effective than the petroleum ether extract. Additionally, MBCs for the aqueous extract were not determined within the 1-4 mg/mL concentration range.

Phytochemical screening using GC-MS
Figure 2 shows the chemical groups identified in the root extracts of P. lanceolata.
 

Tables 3 and 4 present detailed constituent profiles of these extracts.












In the petroleum ether extract, fatty acids and esters were the dominant compounds, accounting for 57.57% of the total composition. The key components included hexadecanoic acid, ethyl ester (16.15%), palmitic acid, methyl ester (5.03%), and 9,12-Octadecadienoic acid, ethyl ester (4.72%). In contrast, the aqueous extract w::as char::acterized by a major peak of 3-methoxy-2,2-dimethyloxirane, comprising 30.78% of the extract.
Both petroleum ether and aqueous extracts contained siloxane compounds, specifically cycloheptasiloxane, tetradecamethyl-, and cyclohexasiloxane, dodecamethyl-, at varying concentrations. Notably, these compounds were more abundant in the aqueous extract (17.61% and 9.02%, respectively) compared to the petroleum ether extract (0.88% and 0.33%). 

Discussion 
P. lanceolata is a medicinal plant valued both traditionally and in modern medicine for its ability to address a wide range of serious health conditions. Researchers have consistently confirmed its biological activities, particularly its antibacterial properties. Various studies have documented different levels of antibacterial effectiveness of P. lanceolata extracts against pathogenic bacteria [15, 19]. Specifically, the antimicrobial activity of the aqueous extract against P. vulgaris has been described as weak or moderate [20]. Similarly, in the current study, the aqueous root extract demonstrated weaker antibacterial activity than the petroleum ether extract. Another study highlighted the potent antibacterial activity of the pure petroleum ether extract from P. lanceolata leaves against pathogenic bacteria [21]. Consistent with these findings, our present research shows that the petroleum ether extract of P. lanceolata roots exhibits significant inhibitory effects on the tested bacterial strains. Our previous study showed the antibacterial effect of the dichloromethane root extract of P. lanceolata against S. paratyphi at a concentration of 100 mg/mL [12]. However, the results of this study align well with earlier research, reinforcing the antibacterial efficacy of P. lanceolata extracts. 
The root extracts of P. lanceolata exhibited distinct chemical profiles depending on the solvent used. The dichloromethane and ethyl acetate extracts were dominated by 1,2-Benzenedicarboxylic acid, mono (2-ethylhexyl) ester, constituting 60.64% and 60.93% of their compositions, respectively. Meanwhile, the butanol extract primarily contained 2-methyl-1-butanol (±)- (17.85%) [12]. Notably, these specific compounds were absent in the petroleum ether and aqueous root extracts analyzed in this study. Comparative analysis revealed similarities between the n-hexane leaf extract of Iraqi P. lanceolata, which contains hydrocarbons, fatty acids, steroids, and terpenoids [22], and the Iranian petroleum ether root extract, which contains some overlapping components. Additionally, the presence of siloxane derivatives (e.g. cycloheptasiloxane and cyclohexasiloxane) in extracts is likely an artifact of gas chromatography column bleeding rather than natural plant constituents [23].
The aqueous root extract of P. lanceolata predominantly contained 3-methoxy-2,2-dimethyloxirane (30.78%), a low-molecular-weight epoxide also identified via GC-MS in methanolic extracts of Cyperus alternifolius (4.29%) [24], ethanolic extracts of Carica papaya L. [25], and ethanol extracts of single-use plastic bags (84.26%) [26]. The widespread occurrence of this compound across various plant and plastic-derived matrices suggests an artifactual origin, potentially arising from extraction solvents, epoxy resin degradation, or laboratory contamination. Therefore, orthogonal validation using NMR or authentic standards is crucial before assigning biological significance in phytochemical analyses [27].
The antibacterial activity of P. lanceolata extracts is attributed to specific antibacterial compounds identified by GC-MS analysis. These compounds include 1,2-benzenedicarboxylic acid, diisooctyl ester [28], gamma.-sitosterol [29], hexadecanoic acid, methyl ester [30], linolenic acid [31], 9-octadecenoic acid (Z)-, methyl ester [31], Z-10-octadecen-1-ol acetate [32], octadecanoic acid [33], 9,17-octadecadienal, (Z)- [34], palmitic acid [35], pentadecanoic acid, ethyl ester [36], Phytol [37], stearic acid [38], trans-vaccenic acid [38], and other compounds.
Compounds, such as oxiranes, silanes, and siloxanes in aqueous extracts, may vary depending on the plant’s geographical origin and environmental factors. However, additional studies are needed to better understand the underlying mechanisms and identify the main bioactive substances responsible for the plant’s therapeutic properties.

Conclusion
Although various researchers have evaluated the antimicrobial activity of P. lanceolata leaves, the current study examined the antimicrobial properties of petroleum ether and aqueous extracts of P. lanceolata roots. Numerous compounds with antibacterial properties have been identified in P. lanceolata, confirming its antibacterial potential. The present research suggests conducting in vivo experiments and clinical evaluations to improve the understanding of the properties of plant roots.

Ethical Considerations
Compliance with ethical guidelines
There were no ethical considerations to be considered in this research.

Funding
This paper was extracted from a PhD Dissertation of Samaneh Rahamouz-Haghighi, approved by the Department of Plant Production and Genetics, Faculty of Agriculture, University of Zanjan and financially supported by, University of Zanjan, Zanjan, Iran (Grant No.: A-12-848-35).

Conflict of interest
The author declared no conflict of interest.

Acknowledgments
The author appreciate the support of University of Zanjan, Zanjan, Iran.

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