Investigation of the Effects of Lavandula stoechas Oil on Neuronal Damage Resembling Epilepsy Induced by Pentylenetatrazol in HT-22 Cells
Patent Information
- Application Number
- TR202600231
- Authority / Receiving Office
- TR · TR
- Patent Type
- Applications
- Current Assignee / Owner
- Filing Date
- 2026-01-08
- Publication Date
- 2026-06-22
Smart Images
Figure 00000008_0000 
Figure 00000008_0001 
Figure 00000008_0002
Abstract
Description
1 TARIFF Lavender (Lavandula stoechas) Oil in HT-22 Cells On Neuronal Damage Such as Epilepsy Induced by Pentylenetatrazol Investigation of its Effect5 METHOD Our study investigated neuronal induced in HT-22 cells by pentylenetetrazol (PTZ). Experimental stages related to investigating its effect against damage, Sivas Cumhuriyet At the University Medical Faculty Research Center, on cell cultures10 This has been done. Experimentally, chemical, biological or physical factors... Determining the viability of exposed cells after exposure, this This is an important step in the research. Regardless of the type of cytotoxicity study being conducted. However, the important thing is to determine the amount of live / dead cells at the end of the study. XTT In addition to not requiring any additional solving process in the test, during the experiment15 It is possible to record the absorbance at various times. This allows... more detailed information about the assessment of viability and more about cell count It contains sensitive information. Cell Lines and Chemicals20 The HT-22 (SCC129) mouse hippocampal neuronal cell line was used in this study. The cell line was sourced from Sigma Aldrich (Missouri, United States). Dulbecco's, a high-glucose product used in cell growth and multiplication, has been developed. Modified Eagle's Medium (DMEM), Fetal Bovine Serum (FBS), L-glutamine, 25 Penicillin / streptomycin (10,000 U / mL), trypsin-EDTA solution, and PTZ Sigma Aldrich Purchased from a company in Missouri, United States. Lavender oil. Purchased from Türkiye. Pentylenetetrazol (PTZ) was used to create an experimental seizure and epilepsy model.30 Pentylenetetrazol is a pharmacological agent used to block the GABAA receptor and chloride. It causes this effect by suppressing the flow. Because of this characteristic... experimentally inducing seizure and post-seizure neuronal damage. It is frequently used in studies. 35 Cell Culture Protocol Under sterile conditions, at 37 °C and 5% CO2, HT-22 neuronal cells in DMEM containing 10% FBS, 5% L-glutamine and 1% penicillin-streptomycin The cells were multiplied in culture medium. When the cells reached 80% density, they underwent passage. It was done and work began after the third passage. The cells are basically four40 They are divided into different groups. These are: Control group: The cells in this group were not treated. 2 PTZ group: Cells in this group were incubated with 60 mM PTZ for 3 hours. KOY + PTZ group: Cells in this group received 100, 50, and 25 µg / mL KOY for 24 hours. After treatment, it was incubated for 3 hours with 60 mM PTZ. (KOY: Karabaş) oil obtained by water distillation of the plant Lavandula stoechas L. KOY group: Cells in this group were treated for 24 hours without subsequent PTZ application. The samples were treated with 100, 50, and 25 µg / mL KOY. GC-MS Method To ensure that plants are standardized in the form of therapeutic preparations, Characterization of the active ingredients, their toxicity10 evaluation, elucidation of the mechanisms of action through scientific research It is necessary, and within the scope of my study, the active components of the lavender plant are also needed. detection is done using one of the advanced technology analysis methods, the chromatographic method. This was done using a working GC-MS instrument. Analysis of the chemical components of lavender oil, Sivas Cumhuriyet University. GC-MS instrument at the Advanced Technology Research and Application Center (CÜTAM) This was carried out using gas chromatography / mass spectroscopy (GC-MS), two It is a combination of powerful analytical techniques. Gas chromatography analyzes the components of a mixture. It separates them. Mass spectroscopy helps in the structural characterization of each component.20 It is possible. Even the identification of very small sample sizes is fast and powerful. It is analyzing. The vast majority (90%) of essential oils consist of terpenic compounds. Terpenic substances include monoterpenes, sesquiterpenes and diterpenes in essential oils. They are found as follows. Oxygenated derivatives formed by the oxidation of terpenes are volatile. It gives the oils their unique scent, taste, and therapeutic properties. Therefore, volatile oils... When studying oil-containing plants, the focus is on the oxygen-containing compounds they contain. Lavender Oil (PAO) and Cell30 After PTZ-Induced Neuronal Damage Impact on Survival: Incubation of HT-22 cells with PTZ for 3 hours resulted in a higher risk compared to the control group. It significantly reduced cell survival (P < 0.001; Figure 1). However, At all doses, KOY was compared with the group treated with PTZ only.35 It has been determined to increase cell survival (P < 0.001; Figure 1). Furthermore, alone... Survival of HT-22 cells was increased by KOY at the administered doses compared to the control. No toxic effect was determined (P > 0.05; Figure 1). In this study, in vitro The effect of lavender oil on neuronal damage caused by neuronal damage has been studied. According to the current literature, this is the first 40 studies conducted in this field regarding lavender oil. It is a study. 3 Assessment of Cell Viability To evaluate cell survival after neuronal damage induced by PTZ. XTT (Biological Industries, Kibbutz) imparts color through mitochondrial enzymes. The Beit-Haemek, Israel test was used. The groups were given the protocol5 mentioned above. After this process, the culture medium was removed and the cells were washed with PBS. Each 100 µl of colorless DMEM was added to the well, and then 50 µl of XTT solution was added on top of it. Incubated for 4 hours. Color change occurs depending on cell viability. microplate reader (Spectrostar Nano, Allmendgrün, Germany) at 450 nm By reading, the cell survival rate of the control group is accepted as 100%, and % Cell10 survival = (Concentration OD / Control OD) × 100 using this formula It has been calculated. Cytoplasmic Calcium Measurement HT-22 Cell Culture Under sterile conditions, at 37 °C and 5% CO2, HT-22 hippocampal neurons were studied. cell line DMEM containing 10% FBS (fetal bovine serum), 1% penicillin-streptomycin The cells were multiplied in cell culture medium. When the cells reached a density of 70-80%... Experiments have begun.20 Oxidative Stress Model and Applications 1 mM hydrogen peroxide (H₂O₂) is applied to cells to induce oxidative stress. Lavender oil (KO) was applied in the experimental groups before H₂O₂ application.25 The specified concentration (100 µg / mL) was added to the cells. The cells were basically four They were divided into different groups. These experimental groups were formed as follows: 1. Control group: The cells in this group were not treated. 2. H₂O₂ (1 mM): Cells were incubated with H₂O₂ for 24 hours.30 3. KO + H₂O₂ group: Cells are treated with H₂O₂ and lavender oil for 24 hours. It was incubated. 4. KO (100 µg / mL) group: Only lavender oil was added to the cells, incubation was performed. receipt. 35 Evaluation of Cell Morphology Cell morphology after the applications is shown in Figures 1 and 2, with 20X magnification. Examined under a light microscope at 40x magnification. Cell density, cell shape, and Changes in cell-cell junctions are morphological indicators of oxidative damage.40 It was evaluated as such. 4 Cytoplasmic Calcium Measurement Intracellular cytoplasmic calcium levels are measured using the calcium-sensitive fluorescent dye Fluo-4. This was determined using AM. After incubating the cells with Fluo-4 AM, the appropriate treatment was applied. It was washed with buffer and the fluorescence signal was measured in a microplate reader. The obtained5 Data were expressed as mean ± standard error (Mean ± ST). Lavender oil and hydrogen peroxide in HT-22 hippocampal neuronal cells. that it maintains cell morphology under the oxidative stress conditions created and It has been shown to significantly reduce the increase in cytoplasmic calcium. The findings,10 Potential of lavender oil against oxidative stress-related calcium dysregulation. This suggests that it may have a neuroprotective effect. Microscopes of all four groups The images are shown in Figure 4. H₂O₂ (1 mM): Significant increase in cytoplasmic Ca²⁺ levels (*P<0.05) (oxidative stress15 (caused by calcium homeostasis disruption) KO + H₂O₂: Ca²⁺ increase was significantly suppressed (#P<0.05) KO (100 µg / mL): Ca²⁺ levels similar to control. Control: Cells are diffuse, spindle-shaped / fibroblast-like morphology, cell-to-cell20 The connections have been preserved. H₂O₂ (1 mM): Causes cell shrinkage, rounding, loss of density, and localized discoloration. Cell detachment → an indicator of oxidative damage. KO + H₂O₂: Cell morphology is significantly preserved compared to the H₂O₂ group, Cell proliferation and integrity have increased.25 COD (100 µg / mL): Similar to control, no toxic effects observed. According to all these findings, lavender oil reduces oxidative stress in cells induced by H₂O₂. It has shown a protective effect against damage. Statistical analysis SPSS 22.0 software was used for statistical evaluation. In all groups The mean ± standard error (Mean ± ST) of the measured cell viability data was calculated. It was evaluated. Since the data showed conformity to a normal distribution, only 35 One-way analysis of variance (ANOVA) was used, and the Tukey test was used post-hoc. The statistical significance level was considered to be P < 0.05. GC-MS Findings 40 In determining the elements in multicomponent mixtures, those present in the gas phase are used. or separation of gasifiable samples by mass chromatography, qualitative and High-performance mass spectrometer system used for quantitative studies. Analysis performed using GC-MS revealed that lavender essential oil contains 37 different types. Component 45 has been identified. The analyses were performed using a method carried out at 180 °C under constant pressure. The resulting chromatogram is shown in Figure 2. Of the 37 compounds identified... Retention times and % relative compositions as major components % Eucalyptol (1,8 cineol) contains 42.31%, while Endo-5,5,6-, a phenolic compound, contains 16.91%. trimethyl-2-norbornanone (oxygenated monoterpene and 9,49 with Camphor (Bicyclo [2.2.1]5 heptan-2-one (1,7,7-trimethyl-) has been identified. It is a naturally occurring organic compound. Eucalyptol (1,8-cineol) is a cyclic ether and a monoterpenoid. Eucalyptol has anti- It has anti-inflammatory, antioxidant, analgesic, antimicrobial and anticancer properties. This has been shown in various studies. Terpenes, such as eucalyptol, enhance drug penetration. These are lipophilic molecules that increase the blood-brain barrier. Eucalyptol can easily cross the blood-brain barrier and10 It can directly affect receptors and enzymes in the brain. Furthermore, eucalyptol... It has been reported in the literature to have an anti-inflammatory effect in neurodegenerative diseases. Camphor, a main component of plant essential oils, has been used in the food and pharmaceutical industries for many years. They are widely used. The active agents in our study are these major compounds. It is estimated that there may be phenolic compounds.15 Conclusion This study, considering the currently available literature, is based on the Karabaş The first in vitro study to investigate the neurological interactions and potential effects of plant oil. This is a study. In our study, lavender oil was found to induce PTZ-induced neuronal activity in neuronal cells. It has been determined that it increases cell survival by preventing damage. Lavender This neuroprotective property of (Lavandula stoechas L.) oil is due to its major25 components. It is thought to be achieved through compounds. Studies are pharmacological. with its repetition and in vitro development, the risks of neurological diseases and it is thought that it may be helpful in reducing symptoms. Therefore The plant extract we used in our study has different effects on neurological systems. Further studies are needed to elucidate their interactions and mechanisms.30 Epileptic seizures and epileptogenesis in Lavandula stoechas L. using in vivo models. after its effects are researched and positive results are obtained, and in the subsequent process Research can also be developed using laboratory animals. 35 Also by gas chromatography / mass spectroscopy (GC-MS) method structural analysis and quantitative determination of the molecules in the essential oil we use Measurements were also carried out. As a result, lavender essential oil was found to be effective against PTZ. It prevents the neuronal damage that occurs and exhibits neuroprotective properties. It has been determined that these effects occur through oxidative and nitrosative systems.40 It is thought that it can achieve this. Lavender oil hydrogen in HT-22 cells. This suppressed the oxidative stress and calcium increase caused by peroxide. It has preserved cellular integrity. These findings suggest that lavender oil protects against oxidative stress. a potential way to support calcium homeostasis against associated neuronal damage This suggests it may be a neuroprotective agent.45 6 Explanation of the Figures Figure 1. Lavender oil (LAO) after PTZ-induced neuronal damage. Effect on cell survival. Data are presented as Mean ± SD. ***P < 0.001 Compared to the PTZ group.5 Figure 2. HT-22 with oxidative damage induced and treated with lavender oil. After staining the cells with Fluo 4-AM, at 20x (a) and 40x (b) magnification Microscope images. Figure 3. HT-22 hippocampus after oxidative damage induced by hydrogen peroxide. The effect of lavender oil on neuronal cytoplasmic calcium with Fluo 4-AM10 Evaluation. Data are presented as Mean ± SD. *P<0.05 compared to the control group. compared. #P<0.05 Compared to H2O2 group. Figure 4. Microscopic images of the four groups studied as a result of the oxidative stress experiment. Figure 5. GC-MS chromatogram
Claims
7 REQUESTS 1. In the treatment or prevention of epilepsy and / or other neuronal diseases Prepared for use, obtained from the Lavandula stoechas plant. a composition containing the extract and / or essential oil obtained.5 2. According to Claim 1, the extract and / or essential oil in question is considered pharmaceutical. characterized by forming a composition with at least one carrier that can be used pharmaceutical compound.
3. According to Claim 1 or 2, the extract in question is mixed with water, ethanol and / or hydroalcoholic beverages. a usage or composition characterized by being obtained using solvents.10 4. According to Claim 3, the form in question is available as tablets, capsules, syrup, cream and / or A composition characterized by being an injectable solution.