The effect of Lavandula Coronopifolia essential oil on the biophysical properties of desensitization and deactivation gating currents in ionotropic receptors

The rising incidence of cancer and the lack of effective therapeutic interventions for many neurological illnesses like Alzheimer's and epilepsy has prompted us to investigate the composition and effects of the Lavandula coronopifolia oil from Palestine on cancer cells and AMPA receptor subunits in the brain due to the vast range of beneficial properties of Lavandula coronopifolia essential oil (EO). GC/MS was used to analyze L. coronopifolia's EO chemistry. EO's cytotoxicity and biophysical effects on AMPA receptors were investigated using MTS and electrophysiological techniques. The GC–MS results revealed that L. coronopifolia EO has a high content of eucalyptol (77.23%), β-pinene (6.93%), and α-pinene (4.95%). The EO showed more significant antiproliferative selectivity activities against HepG2 cancer cell lines than HEK293T cell lines with IC50 values of 58.51 and 133.22 µg/mL, respectively. The EO of L. coronopifolia affected AMPA receptor kinetics (desensitization and deactivation) and preferred homomeric GluA1 and heteromeric GluA1/A2 receptors. These findings indicate the potential therapeutic use of L. coronopifolia EO in the selective treatment of HepG2 cancer cell lines and neurodegenerative diseases.

the Jenin governorate of Palestine; the Lavandula coronopifolia is not protected or regulated in Palestine because it is wild, and there is no legislation prohibiting its research. The plant leaves of Lavandula coronopifolia were collected by WHO standards for evaluating herbal medicines and legislation. All methods followed applicable institutional, national, and international guidelines and legislation. Dr. Nidal Jaradat, a pharmacognosist at An-Najah National University, with a voucher specimen code of pharm-PCT-1367, performed the plant identification and deposition in the Pharmacognosy Laboratory. After the leaves were washed entirely, L. coronopifolia plant EO was extracted by hydro-distillation 24 ; 1 L of distilled water suspended 0.1 kg of fresh aerial parts. The EO was extracted utilizing hydro-distillation with Clevenger apparatus using air pressure at 100 °C for 150 min. Calcium carbonate was utilized in the chemical purification procedure to preserve the L. coronopifolia EO, held at 2-8 °C until further usage, and the yield was 2.15% of the total weight.  [28][29][30][31] . Cells rested for 36 h before electrophysiology recordings or stereomicroscope imaging by replating them on coverslips coated with Laminin (1 mg/mL; Sigma, Germany), cells exhibiting the most fluorescence were selected. Whole-cell (patch-clamp) current recordings were collected using Integrated Patch Clamp Amplifiers with Data Acquisition System (IPA, Sutter Instruments, Novato, CA) and a rapid solution exchange system that was achieved by using a piezoelectric translator (Automate Scientific, Berkeley, CA) controlling a two-barrel theta glass pipette. One barrel contained the external (wash solution), and the other contained the L. coronopifolia EO solution with added glutamate (10 mM). The extracellular solution contained 2.8 mM KCl, 150 mM NaCl, 2 mM CaCl 2 , 0.5 mM MgCl 2 , and 10 mM HEPES, all of which had been adjusted to pH 7.4 using NaOH. Borosilicate glass was used to fabricate patch electrodes; the pipette solution was filled with 110 mM CsF, 30 mM CsCl, 4 mM NaCl, 0.5 mM CaCl 2 , 10 mM EGTA, and 10 mM HEPES. It was adjusted to pH 7.2 using CsOH, and the electrode resistance was 2-4 MΩ. The timing and solution exchange rate was calculated from junction potentials at the open tip of the patch pipette after recordings and were generally between 200 and 300 us (10-90% rise time). Two exponentials fitting the current decline from 90 to 95 percent of the peak to the baseline current were used to calculate the time constants for deactivation (τ w deact) and desensitization (τ w des). The weighted tau (τ w ) was derived as τ w = (τf x af) + (τs x as), where af and as are the amplitudes of the fast (τf) and slow (τs) exponential components, respectively. We measured the currents of deactivation and desensitization using 10 mM of agonist (glutamate) for 1 ms and 500 ms, respectively. At − 60 mV potential, pH 7.4, and room temperature (20-23 °C), electrical current was recorded at a high sampling rate by setting the frequency to 10 kHz, and high-frequency noise was filtered through a low pass filter setting to 2 kHz, digitized by SutterPatch Software v. 1.1.1 (Sutter Instruments). All tests were performed in different cells collected from at least 7-9 independent transfections (separated in time). We employed Igor Pro7 (Wave Metrics, Inc) for our data analysis. The supplementary material includes recorded whole-cell recordings and complete data analysis (Table S1).

Statistical analysis.
Statistical differences between the groups and the wild type were examined by oneway analysis of variance (ANOVA), and the significance was set as * p < 0.05; ** p < 0.01; ns, not significant, with values of *** p < 0.05 being considered to indicate statistical significance. The number of tested HEK293T cells by the Lavandula Coronopifolia oil (n = 8) is presented as mean ± SEM. Concentration-response relationships were fitted as composite curves using GraphPad Prism version 6.01 (GraphPad Software) to the Hill equation.
All results were representative of at least three independent experiments.

Results
Lavandula coronopifolia essential oil components. The GC-MS analysis of L. coronopifolia EO identified sixteen components; Table 1 represents 100 percent of the chromatographic area. Figure 1 shows the separation of the most abundant components of eucalyptol (1,8-cineole), β-pinene, α-pinene, and camphor based on their retention times. The x-axis of the chromatogram would represent time, while the y-axis would represent the intensity of the signals detected by the mass spectrometer. Each peak in the chromatogram would correspond to a specific compound in the sample. The height and area of the peak would be proportional to the concentration of the compound in the sample. In this case, the most abundant compound, eucalyptol (1,8-cineole), would be represented by the largest peak in the chromatogram, followed by smaller peaks for β-pinene, α-pinene, and camphor, which accounted for 77. 23 www.nature.com/scientificreports/ The GC-MS chromatogram could be used to identify the individual components of the essential oil, confirm their presence, and determine their relative concentrations. The most abundant components of the oil were eucalyptol (1,8-cineole), β-pinene, α-pinene, and camphor, Cytotoxicity effects. As demonstrated by the MTS assay results, L. coronopifolia EO has cytotoxic effects against breast cancer (MCF-7), hepatocellular carcinoma (Hep3B & HepG2), and cervical cancer (HeLa) tumor cells in a dose-dependent manner. The cell inhibition percentage is presented in Figure S1 besides the IC 50 values, which shows the extent to which a substance or treatment reduces the growth or viability of cells, expressed as a percentage of the control cells not treated with the substance or treatment. However, L. coronopifolia EO showed the best cytotoxic effect against HepG2 cells with an IC 50 value equal to 58.51 ± 2.23 µg/mL, as HepG2 cells were strongly affected by several compounds such as Jugl anthraquinone C derived from Juglans mandshurica, Anethum graveolens EO, and other different natural ones [32][33][34] . This EO was 2-folds more selective to the HepG2 cancer cell line in comparison with the HEK293T cell line, while the selectivity ratio was reduced on the other cell lines (Hep3B, HeLa, and MCF-7) because the IC 50 values were 489.22 ± 1.89, 444.77 ± 2.4 and > 500 µg/ mL for Hep3B, HeLa, and MCF-7 cancer cell lines respectively. Our objective was achieved effectively using observing the apparent inhibitory impact while avoiding the induction of cellular demise or the attainment of saturation. The observed differences in IC 50 values across various cancer cell lines and HEK293T cells offer significant insights into the inhibitory characteristics of Lavandula Coronopifolia Essential Oil. This data holds significance as it aids in the identification of particular cancer subtypes that may be more susceptible to targeted treatment with the essential oil. The evaluation of IC 50 values in diverse cancer cell lines and normal cells contributes to comprehending essential oils' relative potency and selectivity based on their composition. The observations mentioned above have prospective ramifications for precision cancer treatments and the biophysical characteristics of AMPA receptors, opening avenues for additional research endeavors.

Lavandula coronopifolia did not possess an inhibition effect on AMPAR subunits' cell currents.
The whole-cell patch-clamp technique was used to investigate transfected cells' current changes to evaluate if L. coronopifolia EO inhibits homomeric and heteromeric AMPAR-subunits (i.e., GluA1 and GluA1/ A2, GluA2 and GluA2/A3). Glutamate (10 mM) was first administered to the cell for 500 ms to collect evoked current measurements, and then the cells were exposed to the L. coronopifolia EO solution. Before exposure to the L. coronopifolia EO, the current values were represented by A, whereas those following exposures to the oil were represented by A I (all the data analyses are shown in Table S1). Across all the investigated AMPA-type subunits, there was a slight reduction in current in amplitudes (A I ) (Table S1); however, this decrease in currents in all tested AMPAR subunits was insufficient to be considered AMPA receptor inhibition since the A/A I ratio was less than twofold. The L. coronopifolia EO dropped all homomeric and heteromeric subunits' amplitude almost onefold (Fig. 2).
The Lavandula coronopifolia essential oil affects AMPAR biophysical gating properties. The next step was to ascertain the efficacy of L. coronopifolia EO on AMPARs biophysical gating properties to determine pharmacological potencies. One potential therapeutic strategy is the desensitization and deactivation of AMPARs to alleviate persistent excitatory AMPAR activity (Table S1). AMPARs become desensitized when HEK293T cells are exposed to glutamate for 500 ms. Our findings indicate that L. coronopifolia EO has affected the tested subunits since post-L. coronopifolia EO lowered tau (τ w des) values by about one-fold (Fig. 3). When HEK293T cells were treated with L. coronopifolia EO, the desensitization rate of GluA1 decreased by a factor greater than one. Furthermore, the L. coronopifolia EO impact is not coupled in homomeric or heteromeric subunits since they are affected almost the same way (Fig. 3).
The deactivation value (τ w deact) for the GluA1 receptor, on the other hand, increased approximately by twofold following the application of the L. coronopifolia EO, whereas GluA1/A2, GluA2, and GluA2/A3 values increased around onefold (Fig. 4). The EO of L. coronopifolia was generally effective in influencing desensitization and deactivation processes.

Discussion
This research analyzed the chemical composition of Lavandula coronopifolia essential oil, as well as its cytotoxic effects on several cancer cell lines and impact on AMPA receptor subunit cell currents. The pharmacological activities of Lavandula coronopifolia EO's chemical components that have been investigated in this research, eucalyptol, β-pinene, α-pinene, and camphor, are well-known for their antibacterial 35 , antifungal 36 , and antiinflammatory properties 37 . These components, along with the oxygenated monoterpenoids and monoterpene hydrocarbons commonly found in lavender essential oils, have been found to possess various other pharmacological activities, such as antioxidants 38 , antimicrobial, and anticancer effects. In particular, recent studies have shown that Lavandula coronopifolia EO has potent cytotoxic effects against various cancer cell lines, including MCF-7, HeLa, HepG2, and Hep3B. Notably, the IC 50 value of 58.51 ± 2.23 µg/mL for HepG2 cells was lower than that reported for other natural compounds with known anticancer activity, such as Jugl anthraquinone C derived from Juglans mandshurica and Anethum graveolens EO 32 . Moreover, the selectivity ratio was found to be higher for HepG2 cells compared to other cancer cell lines, suggesting that Lavandula coronopifolia EO may hold promise as a selective anticancer agent.
Interestingly, the chemical composition of Lavandula coronopifolia EO can vary depending on a range of factors, such as geographical origin, temperature, relative humidity conditions, soil, genetics, and degree of maturity 39 40 . In contrast to our findings, Messaoud et al. reported that transocimene (26.9%), carvacrol (18.5%), bisabolene (13.1%), and myrcene (7.5%) were the major components of the EO of L. coronopifolia aerial plant parts (leaves and flowers) from Tunisia and that monoterpene hydrocarbons (46.2%) were the most abundant group, followed by oxygenated monoterpenes 38 . Similarly, Hassan et al. studied the L. coronopifolia EO from Saudi Arabia and identified phenol-2-amino-4,6-bis (1,1-dimethyl ethyl) (51.18%) as the main constituent. These findings highlight the variability of Lavandula coronopifolia EO's chemical composition and underscore the importance of considering these factors when assessing its pharmacological properties 8 .
Overall, the combination of potent pharmacological effects and varied chemical composition of Lavandula coronopifolia EO makes it an intriguing target for future research and therapeutic use. A future study might concentrate on discovering the precise chemical components responsible for its anticancer potential, as well as finding the best cultivation and extraction conditions to maximize yield and potency.
AMPARs are abundant on dendritic spines' postsynaptic membranes, are highly active, and shuttle in and out of synapses. When glutamate attaches, AMPARs are activated, opening the pore and enabling the entry of Na + ions (together with K + efflux) to depolarize the postsynaptic compartment 41,42 . Ca 2+ influx is also facilitated by AMPARs, which has significant consequences for plasticity via activating Ca 2+ -dependent signaling systems 43 . AMPAR plasticity dysregulation has been linked to various clinical conditions, including Alzheimer's disease, autism spectrum disorders, Parkinson's disease, epilepsy, amyotrophic lateral sclerosis (ALS), ischemia, and drug addiction 44 . AMPARs overactivation is potently excitotoxic, causing immediate or delayed neurotoxicity, and has significant implications for mental health 45 .  Figure (a) shows the whole-cell recordings of amplitudes (pA) obtained from HEK293Texpressing AMPAR-type subunits (i.e., GluA1, GluA1/A2, GluA2, and GluA2/A3), conducted at − 60 mV, pH 7.4, and 22 °C after treating the cell with 10 mM glutamate (blue) alone, and Glu with a fixed concentration of 120 μM of Lavandula coronopifolia oil (red) for 500 ms. Lavandula coronopifolia oil concentration was chosen for its highest effect without affecting the health of the cells. Figures (b, c, d,) and e show the A/A I ratio, where A represents the current generated by glutamate alone, and A I represents the current caused by glutamate + Lavandula coronopifolia oil. Data shown are mean ± SEM; n = 8 (number of patch cells in the wholecell configuration). Significance was calculated using one-way ANOVA, ns, not significant. www.nature.com/scientificreports/ This study tested the L. coronopifolia EO on homomeric and heteromeric subunits. Heterotetramers of AMPARs are the most common form of brain receptors, considerably increasing the number of functional subtypes. Homomers of AMPARs are possible, although favored heteromers of GluA1-GluA4 in different combinations are preferred 46 . The GluA2 subunit is found in many AMPA receptor complexes, limiting Ca 2+ permeability and low single-channel conductance values 47 . Besides, GluA1-containing AMPA receptors will undoubtedly shed light on novel therapeutic strategies for treating dementia and age-related cognitive disorders of Alzheimer's.
It was essential to investigate and comprehend the biophysical gating properties of AMPA receptors to treat the diseases stated. AMPAR measures are empirical measures of current decay from the active state to the deactivation of AMPARs. The measured decay of current following glutamate removal after a short stimulation time determines deactivation 48 . At the same time, desensitization is a kinetic property of receptors in which channels become liganded but closed (desensitized) over a predictable time. Auxiliary subunits and RNA splicing control the degree of desensitization, which has essential physiological roles of glutamate depending on the kinetics of glutamate in the synaptic cleft and is involved in protecting neurons from the neurotoxic effect 49 . Calcium influx via AMPARs is uncontrolled when AMPAR subunit composition, function, or desensitization kinetics are impaired; downstream pathways are overactivated 50 . Subsequently, managing deactivation and desensitization, essential in creating the synaptic response, could contribute to the development of cancer treatments.

Conclusion
Significant amounts of eucalyptol, β-pinene, and α-pinene were discovered in the EO extracted from the leaves of L. coronopifolia. In vitro, cytotoxicity studies revealed that the EO was potentially cytotoxic, notably in HepG2 cancer cells, and inhibited these cancer cell lines twice as selectively as HEK293T cell lines. L. coronopifolia EO also displayed neuroprotective effects, showing its future potential as a reliable source of phytopharmaceuticals. Preclinical studies on L. coronopifolia EO would be required to establish its safety and effectiveness, but the findings reported here are intriguing and suggest promising possible future applications. . Lavandula coronopifolia effect on AMPAR subunits desensitization rate. Lavandula coronopifolia oil modifies AMPARs desensitization time (τ w des) from HEK293T cells expressing AMPAR-type subunits (GluA1, GluA1/A2, GluA2, and GluA2/A3) upon 500 ms. Figure (a) show the whole-cell current recording that was conducted by exposing AMPAR-type subunits to glutamate (Glu) alone (10 mM) (black) or Glu with Lavandula coronopifolia oil at a fixed concentration of 120 μM (red) . Figures (b, c, d,) and e show the traces obtained in the presence (red) and absence (black) of Lavandula coronopifolia oil. G represents the 10 mM glutamate used in the experiment, noted above the current trace. Data are shown as mean ± SEM; n = 8 (number of patch cells in the whole-cell configuration). Significance (one-way ANOVA): * p < 0.05; ** p < 0.01; ns, not significant. . Lavandula coronopifolia effect on AMPAR subunits deactivation rate. Lavandula coronopifolia oil modifies AMPARs deactivation time (τ w deact) from HEK293T cells expressing AMPAR-type subunits (GluA1, GluA1/A2, GluA2, and GluA2/A3) upon 1 ms. Figure (a) show the whole-cell current recording that was conducted by exposing AMPAR-type subunits to glutamate (Glu) alone (10 mM) (black) or Glu with Lavandula coronopifolia oil at a fixed concentration of 120 μM (red) . Figures (b, c, d,) and e show the traces obtained in the presence (red) and absence (black) of Lavandula coronopifolia oil. G represents the 10 mM glutamate used in the experiment, noted above the current trace. Data are shown as mean ± SEM; n = 8 (number of patch cells in the whole-cell configuration). Significance (one-way ANOVA): * p < 0.05; ** p < 0.01; ns, not significant.