Phytochemical Profiling, Molecular Docking, and ER Stress‐Mediated Apoptosis Induced by Plantago loeflingii L. Essential Oil in Ovarian Adenocarcinoma Cells

ABSTRACT

Plantago loeflingii L. (Plantaginaceae) is traditionally used for wound healing and tumor treatment, yet its cellular effects remain unexplored. This study provides the first integrated evaluation of its essential oil (PLA‐EO), including chemical composition, cytotoxicity, mechanistic assays, and molecular docking. Hydrodistillation followed by GC–MS/GC–FID identified 53 constituents (98.33%), dominated by linalool (23.81%), palmitic acid (15.34%), limonene (10.54%), thymol (8.51%), and α‐terpineol (5.23%). PLA‐EO selectively inhibited ovarian adenocarcinoma BG‐1 cells (IC₅₀ = 5.67 ± 0.6 µg/mL) while sparing MCF7, A549, and normal cells. Mechanistic assays revealed ER stress‐mediated apoptosis, as indicated by ER expansion, GRP78 upregulation, Ca^{2 +} overload, and caspase‐3 activation. To explore upstream triggers, molecular docking was performed against the EGFR kinase domain (PDB: 3W2S). The docking protocol was validated by redocking (RMSD = 0.98 Å), and erlotinib (−8.96 kcal/mol) reproduced known interactions with ASP855, ALA859, and PHE723. Palmitic acid (−9.05 kcal/mol) interacted with ARG836 via hydrogen bonding and hydrophobic contacts, while linalool (−7.17 kcal/mol) engaged ASP855 and PHE723. These interactions suggest possible EGFR modulation, though activity likely reflects synergistic contributions from multiple PLA‐EO components. In conclusion, PLA‐EO exhibits selective cytotoxicity in ovarian cancer cells via ER stress pathways, supported by in silico evidence of EGFR targeting. The combined phytochemical, cellular, and docking results highlight P. loeflingii as a promising source of bioactive essential oils, warranting further in vivo validation.

1. Introduction

The Kurdistan region is rich in medicinal plants that have been used for generations in traditional medicine to treat various ailments. However, most of these plants have not undergone scientific evaluation to validate their therapeutic potential [1]. This presents a significant opportunity to extract bioactive compounds with potential pharmacological applications and introduce them to the global medical market as novel treatments [2]. Evidence of herbal medicine as an ancient practice in this area is reflected in the discovery of the Shanidar IV Neanderthal burial site, where flowers were used in ceremonial interments [3]. This discovery underscores the historical significance of plant use in the mountains and plains of Kurdistan [4]. Despite this heritage and widespread use among Kurdish tribes, scientific studies on the region's ethnopharmacology are still limited [5] (Figure 1).

FIGURE 1.

(A, B) Kurdish vendors offering traditional medicinal remedies. (C) A Traditional healer engaged in the collection of Plantago loeflingii (photographs captured by H.I.M.A.).

Ovarian cancer remains one of the leading causes of gynecological cancer mortality worldwide, with late‐stage diagnosis and chemoresistance contributing to poor prognosis [6]. Conventional chemotherapy, such as cisplatin, is effective but often limited by severe side effects and the emergence of resistant tumor populations, highlighting the need for natural products with novel mechanisms of action and improved safety profiles [7]. Essential oils (EOs), complex mixtures of volatile secondary metabolites, have received increasing attention due to their diverse biological activities, including antimicrobial, antioxidant, and anticancer properties [8]. However, the chemical composition of EOs is highly variable and can be influenced by multiple factors, such as cultivar, harvest time, and extraction method [9]; hydrodistillation time [10]; and geographical origin [11]. This variability underscores the importance of detailed phytochemical characterization as a prerequisite for correlating chemical profiles with biological activity. The anticancer effects of EOs often result from synergistic interactions between major and minor constituents [12], acting through diverse mechanisms including mitochondrial dysfunction [13], and endoplasmic reticulum (ER) stress [14].

ER stress has emerged as a key pathway by which natural compounds induce apoptosis. Disturbances in ER function cause accumulation of misfolded proteins and activation of the unfolded protein response (UPR). Prolonged stress can shift the UPR toward apoptosis, characterized by upregulation of GRP78, disruption of Ca²⁺ homeostasis, and activation of caspase‐3 [15, 16]. In ovarian cancer, epidermal growth factor receptor (EGFR) is frequently overexpressed and associated with proliferation, survival, and therapy resistance [17, 18]. Crosstalk between GRP78 and EGFR further links ER stress to oncogenic signaling, as GRP78 modulates EGFR expression and downstream survival pathways [19].

Calcium signaling plays a pivotal role in ER‐mitochondria crosstalk during apoptosis [20]. Loss of calcium homeostasis leads to mitochondrial Ca²⁺ overload, dissipation of mitochondrial membrane potential, release of cytochrome c, and caspase‐3 activation [20–22, 23]. These mechanisms are common features of apoptosis induced by plant‐derived natural products [13, 24].

Plantago loeflingii L. (Plantaginaceae), traditionally used in the Kurdistan region for wound healing and tumor therapy [25], remains unexplored with respect to the composition and anticancer activity of its EO (PLA‐EO). Reports from related Plantago species suggest the presence of bioactive volatile compounds, positioning P. loeflingii as a potential but unstudied source of anticancer agents.

Here, we present the first comprehensive investigation of PLA‐EO, integrating phytochemical profiling, cytotoxicity assays, mechanistic studies, and molecular docking. Gas chromatography–mass spectrometry (GC–MS) and gas chromatography–flame ionization detection (GC–FID) were used to characterize the chemical composition. Cytotoxicity was assessed against ovarian adenocarcinoma BG‐1 cells, with cisplatin as a reference. Mechanistic assays focused on ER stress‐mediated apoptosis, while docking simulations examined the interaction of two abundant constituents, linalool (a monoterpene alcohol with established anticancer activity) and palmitic acid (a long‐chain fatty acid with potential modulatory effects on cancer metabolism), with the EGFR kinase domain.

This integrated chemical, biological, and computational approach provides novel insights into the anticancer potential of PLA‐EO and establishes a foundation for its further evaluation as a complementary therapeutic candidate (Figure 2).

FIGURE 2.

(A, B) Plantago loeflingii L.; (C) Zrarati region, the collection site of the plant (photographs by H.I.M.A.).

2. Results and Discussion

2.1. Results

2.1.1. Physical Properties of the EOs

The EOs extracted from the aerial parts of P. loeflingii were obtained as yellowish oils with a yield of 0.11 ± 0.02% (w/w) based on the dry weight of the plant material. The specific optical rotation of the PLA‐EO was determined as [α]²⁰ _D +1.91 (c = 0.1, CH₂Cl₂), indicating slight optical activity. Furthermore, the relative density of the oil was measured as 0.78 ± 0.03 g/mL, reflecting its low‐density characteristics. These results provide insights into the physicochemical properties of the PLA‐EO, which may influence its bioactivity and potential applications.

2.1.2. Chemical Composition of the EOs

GC–MS and GC–FID analyses identified 53 components within the EO, listed in the order of their elution. These components are expressed as percentages of the total oil composition. The qualitative and quantitative data obtained from the analysis on the HP‐5 column are summarized in Table 1 and (Figures 3 and 4).

TABLE 1.

Chemical composition of the essential oils from aerial parts of Plantago loeflingii (PLA‐EO).

Compound name a	LRI (cal) b	LRI (cal) c	% MS matching d	% in the PLA‐EO e  ± SD	Class of compounds
Hexanal	802	801	90	0.81 ± 0.01	Aldehydes
Capronaldehyde	805	803	91	0.77 ± 0.01	Aldehydes
3‐Methylbutanoic acid	837	835	89	0.12 ± 0.12	Carboxylic acids
Ethyl isovalerate	845	847	81	0.07 ± 0.02	Esters
n‐Hexanol	859	861	94	0.91 ± 0.01	Alcohols
Heptanal	902	902	88	0.33 ± 0.05	Aldehydes
2‐Acetylfuran	914	909	93	1.11 ± 0.14	Furan derivatives
α‐Ethylcaproaldehyde	957	945	84	0.29 ± 0.03	Aldehydes
Benzaldehyde	961	952	92	0.23 ± 0.06	Aldehydes
β‐Pinene	972	974	91	3.91 ± 0.02	Monoterpene hydrocarbons
Octen‐2‐ol	982	980	70	0.31 ± 0.01	Alcohols
2‐Amylfuran	991	996	88	0.51 ± 0.11	Furan derivatives
n‐Octanal	1001	1003	85	0.91 ± 0.07	Aldehydes
Limonene	1020	1022	94	10.54 ± 0.10	Monoterpene hydrocarbons
Hex‐2‐enoic acid	1027	1034	79	Tr	Carboxylic acids
Phenylacetaldehyde	1048	1072	83	0.11 ± 0.02	Aldehydes
cis‐Linalool oxide	1071	1067	94	1.71 ± 0.03	Oxygenated monoterpenoids
trans‐Linalool oxide	1079	1082	79	0.62 ± 0.03	Oxygenated monoterpenoids
Linalool	1099	1097	90	23.81 ± 0.04	Oxygenated monoterpenoids
Nonanal	1107	1101	84	0.17 ± 0.03	Aldehydes
(E,Z)‐2,6‐Nonadienal	1155	1156	88	Tr	Aldehydes
(E)‐2‐Nonenal	1162	1161	73	0.05 ± 0.01	Aldehydes
Octanoic acid	1169	1166	84	1.22 ± 0.13	Carboxylic acids
Terpinen‐4‐ol	1175	1174	98	2.17 ± 0.04	Oxygenated monoterpenoids
α‐Terpineol	1188	1186	94	5.23 ± 0.15	Oxygenated monoterpenoids
Safranal	1200	1201	72	0.29 ± 0.02	Miscellaneous aromatic derivatives
Decanal	1207	1203	90	1.01 ± 0.04	Aldehydes
Capraldehyde	1209	1208	84	0.33 ± 0.05	Aldehydes
β‐Cyclocitral	1223	1220	91	0.09 ± 0.01	Miscellaneous aromatic derivatives
(E)‐Anethole	1288	1289	77	Tr	Miscellaneous aromatic derivatives
Thymol	1291	1297	88	8.51 ± 0.02	Oxygenated monoterpenoids
Carvacrol	1293	1299	71	1.40 ± 0.03	Oxygenated monoterpenoids
Undecanal	1306	1305	93	0.14 ± 0.01	Aldehydes
4‐Vinylguaiacol	1313	1317	64	1.56 ± 0.07	Miscellaneous aromatic derivatives
(E,E)‐2,4‐decadienal	1322	1320	73	Tr	Aldehydes
Eugenol	1357	1358	84	3.61 ± 0.03	Oxygenated monoterpenoids
Capric acid	1390	1389	66	0.67 ± 0.03	Carboxylic acids
Dodecanal	1409	1410	90	0.18 ± 0.05	Aldehydes
Geranylacetone	1451	1459	73	0.03 ± 0.01	Ketones
(E)‐β‐Ionone	1488	1486	79	0.14 ± 0.05	Ketones
Tridecanal	1510	1516	90	0.19 ± 0.02	Aldehydes
β‐Bisabolene	1519	1518	81	0.04 ± 0.01	Sesquiterpene hydrocarbons
α‐Cadinene	1544	1539	69	0.13 ± 0.02	Sesquiterpene hydrocarbons
Nerolidol	1561	1563	85	0.34 ± 0.07	Oxygenated sesquiterpenoids
Fokienol	1594	1598	57	Tr	Oxygenated sesquiterpenoids
Hexadecane	1601	1600	91	0.73 ± 0.11	Hydrocarbons
β‐Eudesmol	1659	1660	84	0.14 ± 0.01	Oxygenated sesquiterpenoids
Heptadecane	1701	1701	90	0.21 ± 0.03	Hydrocarbons
Valerenal	1719	1721	66	0.04 ± 0.01	Aldehydes
Myristic acid	1787	1770	97	1.17 ± 0.14	Carboxylic acids
Palmitic acid	1995	1991	93	15.34 ± 0.11	Carboxylic acids
Linoleic acid	2144	2147	91	1.89 ± 0.23	Carboxylic acids
Linolenic acid	2151	2150	84	4.24 ± 0.17	Carboxylic acids

Abbreviation: Tr, trace amount (mean value below 0.03%).

^a Compounds are listed in order of their elution from a HP‐5 column.

^b Linear retention index on a HP‐5 column, experimentally determined using homologous series of standard C₈–C₄₀ n‐alkanes [26].

^c Linear retention index taken from the literature [27] for nonpolar column.

^d % matching of the experimental mass spectrum with the literature [28].

^e % content ± SD of each component in the corresponding oil, calculated as the mean of three determinations.

FIGURE 3.

Effect of Plantago loeflingii L. essential oils (EOs) extract on BG‐1 cell proliferation and viability. (A) The line graph represents the proliferation level of BG‐1 cells with and without 5.67 µg/mL of the EO at 0, 24, 48, and 72 h incubation times. (B) The line graph represents the percentage of cell viability after 24, 48, and 72 h of incubation with and without 5.67 µg/mL of the EO. Student's t‐test was used for the statistical comparison. The data are expressed in mean ± SEM. Groups were considered statistically significant when **p ≤ 0.01 and ***p ≤ 0.001.

FIGURE 4.

PLA‐EO induces endoplasmic reticulum (ER) stress in BG‐1 ovarian cancer cells. (A) Representative fluorescence images of BG‐1 cells stained with ER‐Tracker Red following treatment with PLA‐EO (5.67 µg/mL) for 12 and 24 h, compared to vehicle‐treated control. (B) Quantification of ER‐Tracker Red fluorescence intensity. Data represent mean ± SD of three independent experiments. Scale bar = 50 µm. Groups were considered statistically significant when **p ≤ 0.01 and ***p ≤ 0.001.

In Table 2, terpenoids identified in the PLA‐EO have been grouped in families and other identified compounds have been divided according to the chemical function. Compounds occurring in traces were not considered.

TABLE 2.

Distribution of the compounds identified in the PLA‐EO.

Compound type (total number in the PLA‐EO)	% in PLA‐EO
Monoterpene hydrocarbons (2)	14.45
Oxygenated monoterpenoids (8)	47.06
Sesquiterpene hydrocarbons (2)	0.17
Oxygenated sesquiterpenoids (3)	0.48
Others
Hydrocarbons (2)	0.94
Aldehydes (17)	5.56
Ketones (2)	0.17
Alcohols (2)	1.22
Carboxylic acids (8)	24.65
Esters (1)	0.07
Furan derivatives (2)	1.62
Miscellaneous aromatic derivatives (4)	1.94
Total identified compounds (53)	98.33

2.1.3. Antiproliferative Activity

The in vitro antiproliferative effects of PLA‐EO were evaluated against multiple cancerous and non‐neoplastic control cell lines, including, MCF7 (human breast cancer), BG‐1 (human ovarian carcinoma), A549 (human adenocarcinoma alveolar basal), and Ishikawa (human endometrial cancer) and HUVEC and PBMC cell lines, using cisplatin (cis‐diamminedichloroplatinum(II)) as a reference drug.

IC₅₀ values (mean ± SD) were determined via MTS assay and calculated by probit analysis (p < 0.05, χ ² test), with PLA‐EOs values expressed in µg/mL and cisplatin in µg /mL. The results showed (Table 3) that PLA‐EO exhibited strong antiproliferative activity against BG‐1 and Ishikawa cells, while MCF7, A549, and control cells (HUVEC and PBMC) required concentrations exceeding 50 µg/mL for significant growth inhibition. The IC₅₀ value for BG‐1 cells was 5.67 ± 0.6 µg/mL, indicating high sensitivity to PLA‐EOs. Treatment with 5.67 ± 0.6 µg/mL resulted in a significant reduction in cell viability before inhibiting proliferation, establishing this concentration as the minimum effective dose for further analysis. Viability, assessed using the trypan blue exclusion assay, decreased as early as 12 h posttreatment and continued to decline at 24, 48, and 72 h (Figure 3B). In parallel, a significant reduction in cell proliferation was observed from 24 h onward, with a marked decrease in cell count, whereas nontreated BG‐1 cells continued to proliferate (Figure 3A). The sustained reduction in viability at all time points confirms the cytotoxic effect of PLA‐EOs on BG‐1 cells.

TABLE 3.

In vitro antiproliferative activity of the essential oils hydrodistilled from aerial parts (PLA‐EO) of Plantago loeflingii.

Sample	MCF7	BG‐1	A549	Ishikawa	HUVE cells	PBM cells
PLA‐EO	> 50	5.67 ± 0.6	> 50	14.22 ± 0.5	> 50	> 50
Cisplatin	5.25 ± 0.12	3.68 ± 0.12	3.96 ± 0.09	3.81 ± 0.06	—	—

2.1.4. Induction of ER Stress in BG‐1 Cells by PLA‐EO

Following the evaluation of PLA‐EO's antiproliferative effects, downstream pathways were examined to investigate the mechanism of action, with a focus on ER biogenesis and stress signaling. BG‐1 cells were treated with PLA‐EO at a defined concentration (5.67 µg/mL) for 12 and 24 h. Vehicle‐treated cells served as the negative control. To assess ER stress, ER‐Tracker Red dye was used. Fluorescence microscopy revealed a marked increase in red fluorescence intensity in PLA‐EO‐treated cells after 12 h, indicating ER expansion or stress. This signal was further amplified at 24 h posttreatment compared to vehicle‐treated controls (Figure 4A,4B).

2.1.5. Upregulation of GRP78 Expression in BG‐1 Cells by PLA‐EO

To confirm ER stress induction, the expression of the canonical ER chaperone GRP78 (also known as BiP) was examined. Immunofluorescence staining was performed using a GRP78‐specific antibody on cells treated with PLA‐EO for 12 and 24 h. At 12 h, PLA‐EO‐treated cells exhibited increased green fluorescence localized to the cytoplasm, consistent with upregulated GRP78 expression. At 24 h, GRP78 levels were further elevated, showing a strong signal intensity indicative of persistent ER stress. H₂O₂ (100 µM) was used as a positive control for ER stress induction and produced a similar expression pattern (Figure 5A,5B).

FIGURE 5.

PLA‐EO upregulates GRP78 expression as a marker of ER stress in BG‐1 cells. (A) Immunofluorescence images of BG‐1 cells treated with PLA‐EO (5.67 µg/mL) for 12 and 24 h, stained with anti‐GRP78 antibody (green). Nuclei were counterstained with DAPI (blue). GRP78 expression increased in a time‐dependent manner. (B) Quantitative analysis of GRP78 fluorescence intensity (*p < 0.05). (C) Western blot analysis of GRP78 protein levels after 24 h treatment with PLA‐EO and H₂O₂ (100 µM, positive control). β‐Actin was used as a loading control.

Western blot analysis confirmed the immunofluorescence findings. GRP78 protein levels were significantly increased at 24 h in both PLA‐EO‐ and H₂O₂‐treated cells compared to vehicle‐treated controls (Figure 5C). Densitometric quantification showed a substantial elevation in GRP78 expression, supporting the hypothesis that PLA‐EO induces ER stress in BG‐1 cells.

2.1.6. PLA‐EO Induces Intracellular Calcium Overload in BG‐1 Cells

To evaluate whether PLA‐EO triggers ER stress via calcium dysregulation, BG‐1 cells were treated with PLA‐EO at 5.67 µg/mL for 12 and 24 h. Cytosolic calcium levels were assessed using the calcium‐sensitive fluorescent dye Cal520. Compared to untreated controls, PLA‐EO‐treated cells showed a marked increase in cytosolic calcium at both time points (Figure 6). The fluorescence signal was significantly elevated after 12 h and further intensified at 24 h, indicating progressive calcium accumulation. Quantitative analysis confirmed the increase in calcium levels was statistically significant. These findings suggest that intracellular calcium overload contributes to PLA‐EO‐induced ER stress in BG‐1 cells.

FIGURE 6.

PLA‐EO induces intracellular Ca²⁺ overload in BG‐1 cells. Cal520 green fluorescence intensity at 12 and 24 h in BG‐1 cells nontreated (ctrl) and treated with PLA‐EO.

2.1.7. PLA‐EO Treatment Induces Caspase‐3 Activation in BG‐1 Cells

Caspase‐3 activation was assessed to evaluate the apoptotic effect of PLA‐EO treatment in BG‐1 cells. Caspase‐3 is a critical executioner enzyme in the apoptotic pathway, and its activation is a key indicator of apoptosis. Caspase‐3 activation in BG‐1 cells was measured 24 h after treatment with PLA‐EO, H₂O₂ (positive control), and untreated control. PLA‐EO treatment resulted in a significant increase in caspase‐3 activity, as indicated by a marked increase in fluorescence intensity, consistent with apoptotic signaling. H₂O₂ treatment also induced significant caspase‐3 activation, producing a fluorescence intensity comparable to PLA‐EO treatment, confirming the effectiveness of H₂O₂ as a positive control. Untreated BG‐1 cells showed minimal fluorescence, indicating low baseline caspase‐3 activation. These results indicate that both PLA‐EO induce caspase‐3 activation in BG‐1 cells, supporting the involvement of apoptosis in these treatments (Figure 7).

FIGURE 7.

PLA‐EO induces caspase‐3 activation in BG‐1 cells. Caspase‐3 green fluorescence intensity was measured after 24 h in BG‐1 cells nontreated (ctrl) and treated with PLA‐EO and H₂O₂ (100 µM).

2.1.8. EGFR as a Molecular Target of PLA‐EO: Insights From Molecular Docking

To investigate potential molecular targets of PLA‐EO, docking simulations were performed against the EGFR kinase domain (PDB: 3W2S), a key regulator of proliferation and survival pathways (Table 4).

TABLE 4.

Docking energies and molecular interactions of linalool, palmitic acid, and erlotinib with EGFR kinase domain (PDB: 3W2S).

Compounds	Energy score (kcal/mol)	Receptors (interaction type)
Linalool	−7.17	ASP855 (H‐donor), PHE723 (H–π)
Palmitic acid	−9.05	ARG836 (2× H‐acceptor)
Erlotinib (control)	−8.96	ASP855 (H‐donor), ALA859 (H‐acceptor), PHE723 (π–π)

Note: RMSD of redocked co‐crystallized ligand: 0.98 Å, validating the docking protocol.

Abbreviations: ARG836, strong H‐bond acceptor, contributing to palmitic acid's higher docking score; ASP855, hydrogen bond donor/acceptor, stabilizing polar ligand groups; PHE723, aromatic residue forming H─π interactions with linalool.

The docking protocol was validated by redocking the co‐crystallized ligand of 3W2S. The root mean square deviation (RMSD) between the redocked pose and the original crystal structure was 0.98 Å (Figure S2), indicating reliable reproduction of experimental binding modes. Erlotinib, a clinically approved EGFR inhibitor, was used as a positive control. It achieved a docking score of −8.96 kcal/mol (Figure S1), and engaged ASP855 (H‐donor), ALA859 (H‐acceptor), and PHE723 (π─π stacking), consistent with known interaction patterns. This further validated the accuracy of the docking protocol.

Two abundant PLA‐EO constituents, linalool and palmitic acid, were selected for docking due to their high abundance and reported bioactivities. Linalool exhibited a docking energy of −7.17 kcal/mol, forming one hydrogen donor interaction with ASP855 and an H─π interaction with PHE723. Palmitic acid achieved a stronger docking score (−9.05 kcal/mol) through two hydrogen acceptor interactions with ARG836 and stabilizing hydrophobic contacts contributed by its long aliphatic chain (Figures 8 and 9).

FIGURE 8.

2D (left) and 3D (right) structure of linalool with interactions with amino acids of the protein.

FIGURE 9.

2D (left) and 3D (right) structure of palmitic acid with interactions with amino acids of the protein.

While palmitic acid demonstrated a numerically lower docking energy than linalool, this does not imply superior biological potency. Instead, these results provide structural insights into possible binding modes. Importantly, linalool is a monoterpene with well‐documented anticancer activity, whereas palmitic acid is a long‐chain fatty acid that may act less specifically. Thus, the docking findings should be interpreted cautiously, emphasizing their role as preliminary computational insights rather than definitive measures of biological activity.

Collectively, the docking results indicate that both linalool and palmitic acid are capable of fitting into the EGFR active site and interacting with key residues (ASP855, ARG836, and PHE723). These observations suggest that EGFR modulation may contribute to the observed cytotoxicity of PLA‐EO in ovarian carcinoma cells, while recognizing that multiple constituents and pathways likely act synergistically.

2.2. Discussion

The PLA‐EO demonstrated selective cytotoxicity against BG‐1 ovarian adenocarcinoma cells, with minimal toxicity toward other cancer cell lines and normal cells. This selectivity suggests that the oil contains bioactive components capable of targeting ovarian cancer‐specific vulnerabilities. Comparable results have been reported for other plant‐derived EOs rich in monoterpenoids, reinforcing the therapeutic potential of these natural mixtures.

A total of 53 components (98.33% of the total oil composition) at varying concentrations were identified and quantified in the PLA‐EO by GC–MS and GC–FID analyses (Table 1). The mass spectrum of most identified peaks matched the corresponding reference compound in the literature [29, 30] for more than 85%. Six constituents, namely, linalool (1) (23.81 ± 0.04%), palmitic acid (2) (15.34 ± 0.11%), limonene (3) (10.54 ± 0.10%), thymol (4) (8.51 ± 0.02%), α‐terpineol (5) (5.23 ± 0.15%), and linolenic acid (6) (4.24 ± 0.17%) (Figure 10) accounted for about 68% of the PLA‐EO content (Table 1).

FIGURE 10.

Chemical structures of main components of the oils hydrodistilled from aerial parts of Plantago loeflingii: linalool (1), palmitic acid (2), limonene (3), thymol (4), α‐terpineol (5), and linolenic acid (6).

PLA‐EO demonstrated the strongest cytotoxic activity against BG‐1 and Ishikawa cells, with BG‐1 cells showing the lowest IC₅₀ value (5.67 ± 0.6 µg/mL). This selective activity was absent in MCF7, A549, and non‐neoplastic cells (HUVEC and PBMC), supporting tumor‐specific selectivity. Furthermore, the antiproliferative effect was time‐dependent, with sustained reductions in viability and proliferation over 72 h.

Mechanistic assays indicated that PLA‐EO induces apoptosis through ER stress‐mediated signaling. Fluorescence microscopy showed marked ER expansion within 12 h, which increased further at 24 h. This phenotype was corroborated by GRP78 upregulation, a canonical ER chaperone. In addition, PLA‐EO treatment caused significant cytosolic Ca²⁺ accumulation at both 12 and 24 h, consistent with ER stress, since Ca²⁺ overload is a hallmark of UPR activation. Sustained Ca²⁺ dysregulation can drive apoptosis via mitochondrial and caspase‐dependent mechanisms. In line with this, PLA‐EO treatment increased caspase‐3 activation after 24 h, confirming engagement of pro‐apoptotic pathways.

While EGFR inhibition is classically associated with proliferation and survival signaling, some reports suggest that EGFR modulation can indirectly contribute to ER stress under specific conditions. To explore this possible upstream mechanism, molecular docking was performed against the EGFR kinase domain (PDB: 3W2S). The docking protocol was validated by redocking the co‐crystallized ligand, which reproduced the crystallographic pose with an RMSD of 0.98 Å. Erlotinib, a clinically validated EGFR inhibitor, was used as a reference and yielded a docking score of −8.96 kcal/mol (Figure S1), engaging ASP855, ALA859, and PHE723, consistent with known binding interactions.

Among the PLA‐EO constituents, palmitic acid showed a docking score of −9.05 kcal/mol, forming two hydrogen acceptor interactions with ARG836 and stabilizing hydrophobic contacts from its aliphatic chain. Linalool docked with an energy of −7.17 kcal/mol, forming an H‐donor interaction with ASP855 and an H─π interaction with PHE723. Although palmitic acid displayed a more favorable docking score, this should not be equated with superior biological potency, as linalool is a well‐documented antiproliferative monoterpene, whereas palmitic acid is a fatty acid with less specific activity. These docking results are therefore best interpreted as structural insights rather than conclusive evidence of activity.

Taken together, our data support a model in which PLA‐EO exerts selective cytotoxicity against ovarian cancer cells via induction of ER stress, with EGFR modulation as a potential, but not exclusive, upstream contributor. More importantly, the effects of PLA‐EO likely arise from the combined and possibly synergistic action of multiple constituents, rather than a single compound–single target relationship.

3. Conclusions

This study provides the first integrated phytochemical, mechanistic, and computational evaluation of PLA‐EO. The oil was dominated by oxygenated monoterpenes, with linalool and palmitic acid as major constituents. PLA‐EO selectively inhibited ovarian adenocarcinoma BG‐1 cells while sparing non‐malignant cells, and mechanistic assays confirmed induction of ER stress‐mediated apoptosis through GRP78 upregulation, Ca²⁺ overload, and caspase‐3 activation. Docking simulations, validated by redocking (RMSD = 0.98 Å) and benchmarked with erlotinib, revealed plausible interactions of linalool and palmitic acid within the EGFR kinase domain, suggesting EGFR modulation as a potential upstream contributor. However, the activity of PLA‐EO is more likely multifactorial, reflecting the combined effects of its diverse constituents. Overall, PLA‐EO emerges as a promising candidate for further investigation in ovarian cancer, with future work needed to isolate active compounds, validate molecular targets, and assess efficacy in vivo.

4. Experimental Section

4.1. Plant Material

The aerial parts of P. loeflingii L. were collected in April 2024 during the flowering season from the Zrarati District, Erbil Province, within the Kurdistan Autonomous Region of Iraq. The geographical location was recorded using GPS coordinates: longitude 36°23′38.54″ N; latitude 44°16′59.53″ E. The plant species was identified by Dr. A. Al‐Khayyat, Professor of Botany at Salahaddin University‐Erbil, Iraq. A voucher specimen (accession number 7248) has been deposited in the Herbarium of Salahaddin University for reference. The freshly harvested aerial parts were air‐dried under shaded conditions with active ventilation at room temperature (20°C–25°C) until achieving a constant weight. The dried material was subsequently ground into a fine powder using a laboratory grinding mill to ensure homogeneity. The powdered plant material was stored in airtight containers at room temperature for subsequent analysis. No experiments involving human participants or animals were conducted in this study.

4.2. Isolation of Volatile Fractions

The volatile fractions were isolated from the air‐dried aerial parts (100 g) of P. loeflingii through hydrodistillation using a conventional Clevenger‐type apparatus for 3 h. The procedure was conducted in triplicate to ensure reproducibility. Following distillation, the resulting distillate was extracted three times with 50 mL of diethyl ether. The combined organic layers were dried over anhydrous sodium sulfate, then concentrated using a rotary evaporator, and further reduced under a gentle stream of nitrogen gas (N₂). The obtained samples were stored at −20°C until further analysis by GC/FID and GC/MS. The density, optical rotation, and yield of the EOs were evaluated using standard protocols as described in the literature [31].

4.3. GC–MS Analysis

GC–MS analyses were carried out using a Thermo Scientific Focus GC system coupled with a DSQ mass spectrometer detector, operating in electron‐impact (EI) ionization mode at 70 eV. Separation was achieved using a nonpolar Agilent J&W HP‐5 fused silica capillary column (30 m × 0.25 mm inner diameter, 0.25‐µm film thickness, 5% phenylmethylpolysiloxane; Agilent Technologies Italia S.p.A., Cernusco sul Naviglio, MI, Italy). Helium (He) was used as the carrier gas at a constant flow rate of 1.0 mL/min.

EO samples were prepared by dissolving the oil in dichloromethane (CH₂Cl₂) to a final concentration of 1 mg/10 mL, and 1 µL of the solution was injected into the system in split mode (split ratio 20:1). The GC oven temperature was initially set at 60°C for 1 min, followed by a temperature ramp of 5°C/min up to 260°C, where it was held for 5 min. The injector and transfer‐line temperatures were both maintained at 250°C, and the acquisition mass range was set to m/z 41–350 amu. Data acquisition and analysis were performed using MSD ChemStation software.

4.4. GC–FID Analysis

GC separations were performed using a PerkinElmer GC 2400 instrument (PerkinElmer Italia S.p.A., Milan, Italy). An Agilent J&W HP‐5 fused silica nonpolar capillary column (5% phenylmethylpolysiloxane, 25 m × 0.32 mm inner diameter, 1.05 µm film thickness; Agilent Technologies Italia S.p.A., Cernusco sul Naviglio, MI, Italy) was employed for the analysis. Helium (He) was utilized as the carrier gas at a constant flow rate of 1.0 mL/min. The injector temperature was set to 250°C, while the FID was maintained at 260°C. The oven temperature program began with an initial hold at 60°C for 3 min, followed by a temperature ramp of 5°C/min until reaching 260°C, where it was held for 1 min. A 1 µL aliquot of each EO solution (1 mg/10 mL in dichloromethane) was manually injected in split mode (split ratio 27:1). The relative composition of each oil component (Table 1) was determined based on the percentage of the corresponding FID peak area relative to the total peak area, without applying a response‐factor correction. Mean percentage abundances and standard deviations were calculated from triplicate analyses for each EO. Data acquisition and processing were performed using HP3398A GC ChemStation software (Hewlett‐Packard, Rev. A.01.01).

4.5. Identification of EO Components

The identification of chemical constituents in the EO (Table 1) was achieved through a comparison of the acquired mass spectra with reference spectra available in the Adams library [28] and NIST 98 library [32]. In addition, identification was corroborated by comparing the experimentally calculated linear retention indices (LRIs) with those reported in the literature. The LRIs were determined using a homologous series of n‐alkanes (C₈–C₄₀) supplied by Sigma‐Aldrich (Milan, Italy), following the method described by van den Dool and Kratz [26]. This approach ensured the accurate characterization of EO components.

4.6. Determination of Antiproliferative Activity (MTS assay)

The MTS assay for evaluating in vitro antiproliferative activity is based on the reduction of the yellow‐colored MTS inner salt [3‐(4,5‐dimethylthiazol‐2‐yl)‐5‐(3‐carboxymethoxyphenyl)‐2‐(4‐sulfophenyl)‐2H‐tetrazolium] by NAD(P)H‐dependent dehydrogenase enzymes in metabolically active cells to a purple formazan salt, which is soluble in cell culture media [29]. The formazan salt has an absorbance maximum near 490 nm. When cells die, they lose the ability to convert MTS into formazan; color formation thus serves as a convenient marker of only the viable (living) cells, and the measure of the absorbance can be directly related to the number of viable cells.

Four human cancer cell lines, breast (MCF7), ovarian (BG‐1), lung (A549), and endometrial (Ishikawa) cancer cell lines, along with two normal cell lines (HUVE and PBM), obtained from ATCC, were used in the test. The cells were cultured in RPMI‐1640 or DMEM/F‐12 medium (Euroclone, S.p.A., Milan, Italy) supplemented with 10% fetal bovine serum (FBS), 100 µg/mL penicillin/streptomycin and 2 mM l‐glutamine (Life Technologies, Milan, Italy) at 37°C under a humidified atmosphere in presence of 5% CO₂, changing the liquid growth medium whenever needed. When a cell culture reached 70% confluence, a small amount of trypsin was added to the medium to separate the cells from the flask; after 3 min of incubation at 37°C, 1 mL of FBS was added to stop the action of trypsin and to avoid degradation of the cell membrane. Subsequently, the cell‐containing medium was transferred into a cell strainer and centrifuged (ALC 4232 Centrifuge) at 1000 rpm for 10 min. The resulting pellet was resuspended in the growth medium (1 mL), and the cells were separated using an automatic pipette and counted using a counting chamber and trypan blue as dye. Quadruple cell samples were grown in 96‐well flat‐bottom microtiter plates (Cellstar, Greiner bio‐one) at a density of 3 × 10⁵ cells/mL of growth medium in each well. After 2 h of incubation, the medium was replaced with 100 µL of test medium (RPMI‐1640, added with 0.005% l‐glutamine, penicillin, and streptomycin), and the microplates were left in the incubator for an additional 24 h. To determine the antiproliferative activity, six concentrations ranging from 1 to 100 µg/mL in DMSO were prepared for PLA‐EO, and cisplatin, used as the cytotoxic reference compound. The medium in the wells was replaced with a solution (100 µL) of increasing sample concentration. Three replicates were performed for each sample dilution. The microplates were then incubated for 24 h in a humidified atmosphere of 5% CO₂ at 37°C; subsequently, the sample‐containing medium was replaced with fresh test medium (100 µL) and 20 µL of MTS tetrazolium reagent (CellTiter 96‐AQueous One Solution Cell Proliferation Assay, Promega Italia S.R.L., Milan, Italy). The plates were incubated for 2 h at 37°C and the extent of MTS reduction was measured spectrophotometrically at 490 nm using a plate reader (BioRAD Model 550 Microplate Reader). Experiments were conducted in triplicate. Cytotoxicity was expressed as the concentration of compound inhibiting cell growth by 50% (IC₅₀). The IC₅₀ values ± SD (µg/mL) for EOs, while for cisplatin (µM/mL) (Table 4), were calculated by probit analysis (p < 0.05, χ ² test) with GraphPad Prism 4 computer program (GraphPad Software, San Diego, CA, USA).

4.7. ER Stress Detection

BG‐1 cells were cultured in four‐chamber glass‐bottom dishes and treated with PLA‐EO at the indicated concentration for defined time points (12 and 24 h). ER integrity was evaluated using ER‐Tracker Red (Thermo Fisher Scientific), a fluorescent probe selective for ER membranes. After treatment, cells were incubated with 1 µM ER‐Tracker in serum‐free medium for 10 min at 37°C in the dark. Following incubation, cells were washed with PBS. Fluorescent signals were immediately observed using a fluorescence microscope (Nikon, Germany) equipped with a TRITC filter set (excitation: 587 nm, emission: 615 nm). All imaging parameters were kept constant across experimental conditions. Fluorescence intensity was quantified using ImageJ software to assess ER expansion or disruption indicative of ER stress.

4.8. Immunocytochemical Staining

BG‐1 cells were grown for 24 h on 15 mm glass coverslips at a density of 5 × 10³. They were treated with PLA‐EO for a further 24 h. The cells were later fixed with 4% PFA in PBS and permeabilized with 0.2% ice‐cold Triton X‐100 (Sigma‐Aldrich). After multiple rinses with PBS, the BG‐1 cells were then incubated in a blocking solution of 1% BSA. Subsequently, the cells were incubated overnight at 4°C with anti‐GRP78 primary antibody (Cat # PA1‐014A) (Thermo Fisher Scientific). The samples were washed with PBS and incubated with the secondary anti‐rabbit‐FITC (Sigma‐Aldrich) diluted 1:500 in the buffer for 1 h at room temperature. Subsequently, the cells were mounted on microscope slides with 20 µL of Vectashield HardSet mounting medium containing DAPI (Novus Biologicals, Milan, Italy) to visualize the nuclei, and the images were captured with a confocal microscope (Nikon, Germany) with a 64× water immersion objective. BiP‐conjugated green fluorescent cells were excited with 543 nm, and the fluorescence was collected with a 530 nm emission filter, while for DAPI detection a 405 nm laser was sued for excitation, and emission was collected between 430 and 470 nm.

4.9. Caspase Activation Detection

BG‐1 cells were seeded onto glass coverslips placed in 24‐well culture plates. Following fixation with 4% paraformaldehyde, cells were permeabilized using 0.2% Triton X‐100 (Sigma‐Aldrich) for 10 min. After overnight incubation at 4°C with a primary antibody targeting cleaved caspase‐3, cells were rinsed with PBS and subsequently exposed to an AlexaFluor 488‐conjugated goat anti‐rabbit IgG secondary antibody (Invitrogen) prepared in 1% goat serum for 1 h at room temperature. Fluorescence signals were then visualized using a Nikon fluorescence microscope (Germany).

4.10. Intracellular Calcium Measurement

Cytosolic free calcium levels in BG‐1 cells were assessed using the calcium‐sensitive fluorescent probe Cal‐520 AM (AAT Bioquest). Cells were seeded on glass‐bottom confocal dishes and allowed to adhere overnight, then incubated with 5 µM Cal‐520 AM diluted in serum‐free RPMI medium containing 0.04% Pluronic F‐127 for 45 min at 37°C in the dark. Subsequently, the cells were washed twice with pre‐warmed PBS and incubated for an additional 30 min at 37°C to allow for complete de‐esterification of the dye. Images were taken using a confocal laser scanning microscope (Nikon, Germany) equipped with a 488 nm excitation laser and a 500–550 nm emission filter. Images were captured using identical settings across all experimental groups. Quantification of fluorescence intensity was performed using ImageJ software. Changes in intracellular calcium were evaluated by comparing mean fluorescence intensities between treated and nontreated BG‐1 cells.

4.11. Molecular Docking Simulation

The crystal structure of the EGFR kinase domain protein receptor (PDB ID: 3W2S), associated with human ovarian carcinoma, was obtained from the Protein Data Bank [18, 30]. Molecular docking simulations of selected constituents were carried out using Molecular Operating Environment (MOE‐Dock 2015.10) software [33, 34]. The docking process retained the top five ligand conformers based on their highest and best scores, indicating favorable interactions with the active site.

4.12. Docking Validation

To ensure the reliability of the molecular docking protocol, validation was performed by re‐docking the co‐crystallized ligand of the EGFR kinase domain (PDB ID: 3W2S) into its binding site. The native ligand was removed from the crystal structure and then redocked using the same docking parameters applied throughout this study. The resulting docking pose was compared with the experimentally determined crystallographic pose deposited in the Protein Data Bank. The calculated RMSD between the docked and crystallographic conformations was 0.98 Å, well within the generally accepted threshold of 2.0 Å for reliable reproduction of binding orientations. This result demonstrates that the employed docking protocol is accurate and robust, providing confidence in the subsequent docking simulations performed with the selected ligands.

4.13. Statistical Analysis

All experiments were performed in triplicate (n = 3). Data are presented as mean ± standard deviation (SD). Statistical significance was determined using one‐way ANOVA followed by Tukey's post hoc test, and Student t‐test. A p < 0.05 was considered statistically significant.

Author Contributions

Hawraz Ibrahim M. Amin: methodology, validation, formal analysis, investigation, data curation, writing – original draft preparation, supervision.

Conflicts of Interest

The author declares no conflicts of interest.

Supporting information

Supporting File 1: cbdv70676‐sup‐0001‐SuppMat.pdf

Data Availability Statement

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