Herbal medicinal products, derived from plants, are widely studied and utilized for their potential to prevent, treat, and promote health. Natural products have gained recognition as valuable supportive therapies in human health, particularly for conditions like cardiovascular disease, cancer, and neurodegenerative diseases. Alzheimer's disease (AD), a prevalent neurodegenerative disorder primarily affecting older individuals and causing dementia, is characterized by the presence of beta-amyloid protein deposits in the brain's hippocampus region [1]. While the exact causes of AD are not fully understood, various factors including age, genetics, lifestyle, heredity, vascular risk factors, head injury, and diet are believed to contribute to its development. [2]

The damage to cholinergic pathways in the brain closely associated with AD has sparked interest in developing drugs targeting these pathways. Acetylcholinesterase inhibitors are commonly prescribed for AD treatment as they help improve cognitive functions like memory and thinking, particularly in patients with mild to moderate AD. Antioxidants are also considered beneficial in AD, reducing the damaging effects of free radicals on brain cells. Herbal medicine has a long history of use in China for treating dementia, with some individual herbs and herbal formulations showing promise in AD treatment. [3].

Salvia officinalis, commonly known as sage, is a herb with a rich history of culinary and medicinal uses. [4] It has been cultivated for centuries due to its numerous beneficial properties. Sage has been valued for its potential digestive, anti-anxiety, anti-inflammatory, and memory-enhancing effects. It is frequently used in cooking, adding a strong, earthy flavor to various dishes. [5]

While sage offers potential health benefits, it is important to exercise caution and avoid excessive consumption, as it may lead to adverse effects. Sage can also interact with certain medications, necessitating consultation with a healthcare professional before use. Further research is required to fully understand the effects and optimal usage of sage for overall health. [6]

The development of new drugs is a complex and costly process, involving multiple stages from identifying therapeutic targets to clinical trials. Molecular docking, an essential component of drug design, plays a crucial role in determining the interaction of small molecules with targeted proteins. [7] Efforts have been made to enhance methods for calculating molecular docking, benefiting both biological research and the pharmaceutical industry. [8]

The research aims to use in silico activity tests to evaluate the inhibition of the three targets (4EY4,2BEG,1IYT).

The aime of this study is to inhibit the enzyme acetylcholinesterase, and the two peptides beta-amyloid 40, beta-amyloid 42, which are responsible for Alzheimer's disease, using rosemarinic acid extracted from the Salvia officinalis plant.

• Hardware

The specification of the computer that is used: Intel® Core(TM) i5-2450M CPU @ 2.50GHz processor (CPU), HD graphics processing unit (GPU), and 8 GB Random Access Memory (RAM) with Windows 10.

2.2 Chemical compound of salvia officinalis

The principal components in the sage oils were 1, 8-cineole, camphor, α-thujone, βthujone, borneol, and viridiflorol(6). The essential oil contains cineole, borneol, and thujone(7).The essential oils (EOs) extracted from the aerial parts of cultivated Salvia officinalis L. and the berries of Schinus molle L. are 1,8-cineole (33.27%), β-thujone (18.40%), α-thujone (13.45%), borneol (7.39%) in S. officinalis oil and α-phellandrene (35.86%), β-phellandrene (29.3%), β-pinene (15.68%), p-cymene (5.43%) and α-pinene (5.22%) in S. molle oil.(8).its leaf contains tannic acid, oleic acid, ursonic acid, ursolic acid, carnosol, carnosic acid, fumaric acid,chlorogenic acid, caffeic acid, niacin, nicotinamide, flavones, flavonoid glycosides, and estrogenic substances (9).phenolic glycosides cispcoumaric acid 4-O-(2′-O-β-d-apiofuranosyl)-β-d-glucopyranoside and trans-p-coumaric acid 4-O-(2′-O-β-dapiofuranosyl)-β-d-glucopyranoside were isolated and identified from Salvia officinalis together with 4-hydroxyacetophenone 4-O-(6′-O-β-d-apiofuranosyl)-β-dglucopyranoside, luteolin 7-O-β-d-glucoside, 7- and 3′-Oβ-d-glucuronide, 6-hydroxyluteolin 7-O-β-d-glucoside and 7-O-glucuronide, and 6,8-di-C-β-d-glucosylapigenin (vicenin-2). The luteolin glucuronides and vicenin-2 were identified as new sage constituents (9).volatile constituents is heterocyclic monoterpene 1,8-cineole (10, 11). [10]. The most abundant phenolic compounds present in the extracts were rosmarinic acid and luteolin-7-glucoside(12).One abietane diterpenoid, 12-O-methyl carnosol (2), was isolated from the leaves of sage (Salvia officinalis L.), together with 11 abietane diterpenoids, 3 apianane terpenoids, 1 anthraquinone, and 8 flavonoids. Antioxidant activity of these compounds along with 4 flavonoids isolated from thyme (Thymus vulgaris L.). [11]

2.3 Drug-likeness prediction

Three filters of the DruLiTo program (Lipinski's rule, Veber rule, and Ghose filter) were used to predict the drug-likeness of the test compound by entering *sdf file format. [12]

• ADMET prediction

Prediction of pharmacokinetics (ADME) and toxicity of the compound from salvia officinalis was done by the pkCSM website (http://biosig.unimelb.edu.au/pkcsm/prediction) with the SMILES format. [12].

• Molecular docking

The structure of gamma PPARα/γ target receptors (PDB ID of acetylcholinesterase: 4EY4, PDB ID of amyloid beta 40:2BEG, PDB ID of beta amyloid 42:1IYT) obtained from the Protein Data Bank (https://www.rcsb.org/) that is shown in Table 1. As for a comparison ligand, Molecular docking is done using AutoDockTools 4.2.6 program. The validation results are indicated by the Root Mean Square Deviation (RMSD) value. Center the grid box using a grid box (40 × 40 × 40). The binding

• Site coordinates of 4EY4 are x = -0.946; y = -52.365; z = 2.83 with spacing per unit 0.375 angstrom.
• Site coordinates of 2BEG are x = 0.385; y = 0.25; z = -8.949 with spacing per unit 0.375 angstrom.
• Site coordinates of 1IYT are x = -0.616; y = -0.065; z = 1.269 with spacing per unit 0.375 angstrom.

 Visualization analysis of protein-ligand interactions was performed with Discovery Studio Visualizer v.21.1.0.20298 from BIOVIA.

• preparation of ligand

The PubChem (https://pubchem.ncbi.nlm.nih.gov) [13] is a free and open resource, which contains information on small molecules and their biological activities. The ligand Rosmarinic acid (PubChem ID: 5281792).

2.7 Pharmacological effect in Alzheimer disease

Efficacy and safety of Salvia officinalis extract using a fixed dose [60 drops/day], in patients with mild to moderate Alzheimer's disease, over a 4-month period. The results of this study indicate the efficacy of S. officinalis extract in the management of mild to moderate Alzheimer's disease. Moreover, S. officinalis may well reduce agitation of patients but these needs to be confirmed. [14]

Table 1. The three major compound from salivia officinalis

Table 2. The three receptors (proteins) preparation

3.1 Drug-likeness prediction

Prediction results of drug-likeness of Rosmarinic acid from Salvia officinalis are shown in table 3.

Figure 1. The Rosmarinic acid molecule .

Table 3. Prediction of Drug likeness with DruLiTo program

• ADMET prediction

The prediction results of the ADMET of Rosmarinic acid are shown in table 4.

Table 4. ADMET prediction

3.3 Convergence of molecular binding of plant (salvia officinalis) compounds arranged with minimal free binding energy (Δ G) and inhibition constant (Ki) with the three proteins

The three tables represents the energy Δ G and inhibition constant Ki

Table 5. The energy delta G and inhibition constant of salvia officinalis and 4EY4

Table 6. The energy delta G and inhibition constant of salvia officinalis and 2BEG

Table 7. The energy delta G and inhibition constant of salvia officinalis and 1IYT

Table 8. Interaction details of the three targets (proteins)

Figure 2.The interaction between rosmarinic acid and 4EY4

Figure 3.The interaction between rosmarinic acid and 2BEG

Figure 4.The interaction between rosmarinic acid and 1IYT

Figure 5.  Interaction between the enzyme acetylcholinesterase and the drug marketed in pharmacies contains the active substance Citicoline.

The RMSD value, or root-mean-square deviation, is a measure of the similarity between the predicted and actual conformations of the ligand-receptor complex. A low RMSD value indicates a good fit between the ligand and receptor, and suggests that the docking protocol is valid. A commonly accepted threshold for RMSD in docking studies is 2 Å. [12]

In the case mentioned, the RMSD value of 1.42 indicates that the docking protocol used in the study is valid, and the predicted conformations of the ligand-receptor complexes are accurate. [12].

The ΔG score, as previously mentioned, is a measure of the thermodynamic favorability of the ligand-receptor interaction. A more negative ΔG score indicates a stronger interaction and higher binding affinity between the ligand and receptor. [12]

Table 4,5 and 6 shows the results of the molecular docking study, which reveal that Rosmarinic acid has the strongest inhibitory activity with a ΔG value of -8.4 kcal/mol, indicating high affinity towards target receptor acetylcholinesterase than with amyloid beta 42 with a ΔG value -6. The Ki value range from 6544 to 18036 which suggest high affinity towards the target receptor.

Hydrogen bonds, hydrophobic interactions, and electrostatic interactions are all important types of interactions that can contribute to the stability of a ligand-protein complex. Hydrogen bonds are formed when a hydrogen atom is shared between two electronegative atoms, such as oxygen or nitrogen. These bonds are relatively weak, but they can contribute significantly to the overall stability of a complex when multiple hydrogen bonds are formed. [12]

Hydrogen bonding also contributes to the affinity of the ligand to the protein/receptor due to the electrostatic interaction between the oxygen or nitrogen atom of the ligand and the hydrogen atom of the protein amino acid can be seen in Table 7 and figure 2,3 and 4 .

The study suggests that Rosmarinic acid with 4EY4 enhances the affinity of the compounds from Salvia officinalis target receptor, indicating greater activity against the target receptor than 2BEG and 1IYT.

 Inhibitor reference ligand

When we studying the inhibitor reference ligand with in silico programs, the linkages between it and the protein (4EY4) .we have got 20% of amino acids (ARG525,LEU386). When we studying the inhibitor reference ligand with in silico programs, the linkages between it and the protein (2BEG) The interaction with inhibitor ligand almost the same with the interaction without it we have got 85% amino acid (MET A:35 , MET B:35, MET C:35 , GLY33, ILC31, GLY33). When we studying the inhibitor reference ligand with in silico programs, the linkages between it and the protein (1IYT) .The interaction with inhibitor ligand almost the same with the interaction without it we have got 78% amino acid ( GLN15, PHE19, VAL12).

Citicoline (active Principle) of (Pharmaceutical Syrup commercial drug )

When studying the active ingredient of Citicoline with in silico programs, the linkages between it and the protein (4EY4) are the following amino-acids we have ( GLN527 , ARG525, GLN525) In the other hand our ligand targeted 42.85 %. When studying the active ingredient of Citicoline with in silico programs, the linkages between it and the protein (2BEG) are the following amino-acids ( GLN15, PHE19, VAL12) In the other hand our ligand targeted targeted 75 %. When studying the active ingredient of Citicoline with in silico programs, the linkages between it and the protein (1IYT) are the following amino-acids (MET35) In the other hand our ligand targeted 20%.

Hotspot  

We have structures of AChE (4EY4) and co-crystallized with different Compounds: The absence or presence of ligands affects the conformation of principal amino acids lining the gorge, e. g., Tyr337 and Tyr341 [173],. We have previously reported significant differences in estimated binding energies for the same compounds with these targets [174]. Here, we show that the target X-ray structure determines whether or not ligand poses reflect mixed-type inhibition.

The hotspot of (4EY4) is: LEU130, VAL132, SER462, ALA434, PRO446, TYR449, GLY323, ARG522,LEU386, VAL33 ,LYS332,ASP131,ARG463,HIS432,ARG463,THR436: results were confirmed by in silico programs, most powerful 4EY4 inhibitor is Rosmarinic acid, highlighting the presence of hydrogen interactions between oxygen and nitrogen atoms found in this inhibitor (our ligand inhibit 5 amino acids), carbonyl oxygen make bound with 4 amino acids: GLY323,ARG522, LEU386, VAL330 and structural nitrogen bound with LYS332 ,which is part of the enzyme active site.

The hotspots of this target (2BEG) are: PHE20, MET35, GLY33, MET35, ILE31.The results were confirmed in silico programs most powerful 2BEG the structures of Amyloid beta-40 (2BEG) different compounds: The presence of rosmarinic ligand effects the conformation of principal amino acids: MET35, GLY33.

The hotspots of this target (1IYT) are: ARG522, LYS332, ALA526, and GLY523.PHE19. We concluded that there are amino acids that also contribute to protein (1IYT) different compound: ARG522, LYS332.

The results of the study indicate that the compounds are from Salvia officinalis. It has potential as drug candidates due to its pharmacokinetic properties and strong binding affinity towards the receptors 4EY4, 2BEG, 1IYT targeted especially acetyclholinesterase 4EY4. Molecular docking results indicate that Rosmarinic acid is the most potent inhibitor with high affinity towards its target receptor. Compliance with Lipinski's rule and Weber's rule supports their potential as drug candidates.

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