Lavender Essential oil Compounds as Antagonists of NMDA Receptor SubunitNR2B: A Molecular Modeling and Experimental Study

Herbal plants are an important source of novel chemical drugs with therapeutic effects. Lavender is one of the most important herbal plants in the world that has many applications in the pharmaceutical and cosmetic industries. Its fresh and dried flowers are also used for cooking or making herbal tea. The lavender essential oil is known to have sedative, anti-flatulent, antidepressant, anti-inflammatory, analgesic, anti-convulsant, anti-parasitic, antioxidant, antimicrobial, cardioprotective, and anti-schistosoma properties and be mast cell degranulation inhibitor and imrpoves digestive disorders. It is used by aromatherapists as the holistic relaxant. Herbal components have been used for prevention and threatment of several diseases such as Alzheimer’s disease that is the most common age-related dementia in the word. The important characteristics of this disease include amyloid-β (Aβ) plaque, hyperphosphorylated tau, neuroinflammation, oxidative stress, and finally neuronal cell death. One of the most important enzymes involving in the conversion of glutamate to glutamine is glutamine synthetase. Its dysfunction leads to overactivation of N‑methyl‑D‑aspartate receptor (NMDAR) resulting in neuronal injury, activation of the mentioned enzymes, hyperphosphorylation of tau protein, cell death, and alzheimer’s disease. NMDAR is a glutamate receptor and ion channel that plays important role in brain physiology and pathology, and has strong therapeutic potential in different pathologic conditions such as NMDAR receptor overactivation. Among the most promising NMDAR-targeting drugs are allosteric inhibitors of GluN2B-containing receptors. One of the important roles of lavender essential oil may be in inhibiting the production of oxygen reactive species (ROS) due to its antioxidant activity. It can activate several signaling cascade associated with hyperphosphorylated tau protein. The present study aims to determine the chemical compounds of extracted lavender essential oil (Lavandula angustifolia Miller) and assess the effect of their antagonists on NMDA receptors in the brain using Gas chromatography mass spectrometry (GC-MS) and computational approaches.

Flowering inflorescences of lavender plant were harvested at full flowering stage from a cultivated farm and were dried in natural conditions for two weeks prior to the essential oil extraction. For the extraction of essential oil, the dried inflorescences (50 g) were distilled by hydro-distillation method for 3 hours using a one-liter full-glass Clevenger apparatus and the procedure proposed by European Pharmacopoeia for determining the oil content (v/w%) with three replications. To find the active constituents of lavender essential oil, a GC-MS was used. The GC-MS was done using an HP-5972 mass spectrometer with electron impact ionization (70eV) coupled with the HP-5890 series II gas chromatograph. Compounds were identified by calculating their retention index relative to n-alkanes (C9-C18) and the data for authentic compounds available in the literature and in our data bank, and also by matching their mass spectrum fragmentation patterns with corresponding data stored in the mass spectra library of the GC-MS data system. Crystal structure of GluN2B receptor was retrieved from Protein Databank (5EWJ.pdb). PyRx 0.8 virtual screening software in AutoDock Vina was used to perform molecular docking studies on the antagonist compounds of NMDAR in lavender. Twenty components that showed the highest frequency during the experiments were selected for molecular docking studies. Input coordinates for these 20 compounds were obtained from the PubChem website. In order to evaluate the binding affinity of these components (as ligands) with the amino terminal domains of the NMDAR subunit gluN2B, the scoring function (kcal/mol) in AutoDock was applied. The coordinates of the grid box were 24.45´29.90´37.61 Å, and the grid center had a dimension of 24.953´1.872´38.007 Å (x, y and z). Discovery studio v.16.1.0 software was used to prepare two-dimensional diagrams of docking model to show different interactions between NMDAR and ligands. In order to evaluate the results of docking, we used the NMDAR antagonist ifenprodil as a control. The interaction between GluN2B and ifenprodil was assessed using Discovery studio. The results of docking were compared with the residues in the active sites of ifenprodil to find the common residues and the best antagonist. Moreover, 20 lead compounds of phytochemicals were used for pharmacokinetic analysis in accordance with Lipinski’s rule of five.

The essential oil obtained from the lavender inflorescence was yellow-green and its yield was 1.37% (v/w). The 41 compounds identified in the essential oil comprised 95.5% of the total essential oil. The main compound was trans-carveol (15.9%), followed by isopulegol (7.7%,), 1,3,8, -p-menthatriene (7.3%) and isoborneol (6.9%). The compounds of lavender essential oil were categories in seven groups including monoterpenes, sesquiterpenes, benzenoids, ketones, benzenes, alcohols, and aldehydes. The highest numbers of compounds was related to monoterpenes (n=22, 67%) followed by benzenoids (n=6, 12.9%) and sesquiterpenes (n=9, 11.6%). Twenty molecular structures of the lavender essential oil compounds were evaluated in SwissADME website. All compounds had a molecular weight of less than 500 Da. They showed H-bond acceptor <2 and H-bond donor <1. The obtained molar refractivity was in a rangeof 45 to 71. The topological polar surface area was in a range of 0 to 27 Å. The relative solubility as the partition coefficient (P) of the substance is traditionally given between octanol-1 and water. Log P in our study was in a range of 2 to 4. Beta-caryophyllene and germacrene-D compounds were not permeable in the blood brain barrier (BBB), while 1,3,8,-p-menthatriene, gamma-3-carene, gamma-terpinene, p-cymene, alpha-pinene, alpha-phellandrene, Limonene, beta-caryophyllene and germacrene-D were absorbed gastrointestinally in a small amount. Others had suitable permeability in the BBB and gastrointestinal absorption. Beta-caryophyllene and caryophyllene epoxide were CYP2C19 inhibitors; gamma-3-carene, alpha-pinene, limonene, beta-caryophyllene and caryophyllene epoxide inhibited CYP2C9; thymol was CYP1A2 inhibitor, and CYP2D6 was inhibited by p-cymene.

Discussion :

The essential oil of lavender plant cultivated in Alborz province, northern Iran has 41 compounds comprising 95.5 % of the total essential oil. The major identified component was trans-carveol followed by isopulegol, 1,3,8,-p-menthatriene, isoborneol and carvacrol acetate, while the amounts of linalool, citral, beta-ionone, eugenol, 1, octen-3-ol and 5-hepten-1-ol were very low. Camphor, Thymol, beta-Thujone and trans-Carveol compounds of lavender essential oil can lead to inhibiting NMDAR and improving learning and memory in neurodegenerative diseases.