Isolation and Molecular Docking Studies of Dihydroimperialine as Butyrylcholinesterase Inhibitor from the Bulbs of Fritillaria Imperialis

Alzheimer’s disease is a progressive disorder that causes the inhibition of neurotransmitter acetylcholine in brain cells. Fritillaria imperials belong to family liliaceae, comprises several steroidal alkaloids. The subsequent extraction of ethanoic extract was subjected to column and thin layer chromatography, which led to the isolation of two steroidal alkaloids, Dihydroimperialine (1) and imperialine (2). Dihydroimperialine (1) exhibited the acetylcholinesterase and butyrylcholinestearse inhibitory activities with IC50 values as (>500 ± 0.23) and (14.40 ± 1.02), respectively. Compound 1 and imperialine (2) inhibited cholinesterase enzymes in a concentration-dependent manner. Additionally, molecular docking studies were also performed to investigate the binding mode of dihydroimperialine (1) with butyrylcholinestearse. The ligand–BChE complex were found to be stabilized by hydrophobic contacts, hydrogen bonding, and π-π stacking between the compounds and amino acid residues. The main object of this study to investigate the acetylcholinesterase and butyrlcholinestrase inhibitory activity from the bulb of F. imperials. This is the first report of the isolation of dihydroimperialine (1) form natural source.


Introduction
Acetylcholinesterase is an enzyme whose primary function is to breakdown of acetylcholine into choline and Acetyl-CoA, which effects on the memory loss and other intellectual properties. The Alzheimer's Disease (AD) is associated with the loss of cholinergic neurons in the brain and the decreased level of ACh [1,2]. Fritillaria imperialis L commonly known as "crown imperial". The plant usually grows in rocky slopes and amongst scrub at attitude of 1000-3000 meters in Turkey and other parts of eastern Europe.
The bulb is known to be poisonous and contains low concentrations of toxic alkaloids. The genus Fritillaria belongs to the family Liliaceae, which is an important steroid alkaloid producing plant family [3]. Up to now, more than 160 alkaloids have been reported from the genus Fritillaria. A few species of Fritillaria are native to Cyprus, Southern Turkey and Iran [4]. Among them F. roylei or F. verticillate (thembergii) commonly known as "Bei-mu" or "Pei-mu" in Chinese and "Bai-mo" in Japanese, has long been known as one of the principal Chinese crude drugs [3,5] having many therapeutic applications [6]. Many species of Fritillaria are traditionally used as herbal remedies in Japan, Turkey and south Asia [7]. F. [11]. Several alkaloids, such as forticine, impericine, impranine, dihydroimpranine, fetisinine have already been reported from this plant, which showed acetylcholinesterase inhibitory activity [7,[12][13][14]. Butyrylcholinesterase (E.C. 3.1.1.8), metabolizes cocaine to the water-soluble inactive compounds ecgonine methyl ester and benzoic acid [15]. The normal level of BChE varies among individuals and is dependent on age, state of health, exposure to environmental toxins and genetic factors. Low levels of BChE are associated with cocaine-induced cardiac and related complications [16]. Furthermore, deficiency of BChE shifts the metabolism of cocaine to norcocaine, a metabolite with anesthetic and cardiotoxic properties and benzoylecgonine, a metabolite that causes vasoconstriction [16,17]. BChE is distinguished from other esterases by a high affinity for cholinesterase and their thio analogs [17]. It has also been found that butyrylcholinesterase inhibition may also be an effective tool for the treatment of AD and related dementias [17]. Availability of the X-ray crystal structure of butyrylcholinesterase with butyrylcholine (PDBcode: 1P0P) prompted us to perform molecular docking studies. To further explore the binding mechanism of compound 1, molecular docking studies were carried out by automated GOLD docking software.

General
Optical rotations were measured on a Jasco DIP-360 digital polarimeter by using 10 cm cell tube. IR spectra were recorded on a Jasco A-302 IR spectrophotometer. I H-NMR spectra were recorded on Bruker 500 MHz NMR spectrometer and chemical shifts were calculated with reference to CDCl 3 (7.25). 13 C-NMR spectra were recorded at 125MHz on a Bruker AM 500MHz NMR spectrometer.
Mass spectra were recorded on a Varian MAT 312 double focusing spectrometer, connected to an IBM-AT compatible PC computer system. Column chromatography was performed on silica gel (70-270 mesh, ASTM, Merck) and flash silica gel (230-400 mesh, ASTM, Merck). TLC was done on percolated TLC cards (Merck) with solvent system of acetone: pet ether: diethylamine (3:6.5:0.5).

Plant Material
The plant material was already explained in our previous publications [12,13]. The extraction and isolation procedure is the same as described in our previous publication [13]. Column

Cholinesterase Inhibition Assay
The AChE and BChE inhibiting activities were measured by spectrophotometric method developed by Ellman [18]. Electric-eel Louis, MO, USA). All the other chemicals were of analytical grade. Standard operational assay protocol has been described previously [19]. All the kinetic experiments were performed in 96-well microtitre-plates by using Spectra Max 340 (Molecular Devices, CA, USA). The rate of the enzymatic reaction [18] was measured by the following equation.

Determination of IC50 Values
The concentration of compounds that inhibited the hydrolysis of substrates (acetylthiocholine and butyrylthiocholine) by 50% (IC 50 ) was determined by monitoring the effect of various concentrations of the compound in the assays on the inhibition values. The IC 50 values (inhibitor conc. that inhibits 50% activity of AChE and BChE) were then calculated using the EZ-Fit Enzyme Kinetics program (Perrella Scientific Inc., Amherst, USA).

Molecular Docking Methods
Pretreatment of Protein. The high-resolution crystal structure of butyrylcholinesterase in complex with butyrylcholine was obtained from the Protein Data Bank (PDB ID: 1P0P) [20]. All hydrogens were first added into the PDB using the SYBYL program package, version 6.9 [21]. Protonation states were set according to the program, based on the likelihood that the residues would be charged at physiological pH, together with their chances to form hydrogen bonds with the surrounding environment. All water coordinates and metal were removed from the protein file.
Important residues, especially in the binding site were visually inspected and corrected according to charge and according to atom and bond type, especially the pH of lysines, arginines, aspartates and glutamates.

Pretreatment of Ligand
The ligand that was selected for the docking studies was drawn using the sketch module of SYBYL. Ligand was inspected and refined to correct stereochemistry and atom and bond type according to GOLD specifications. In order to obtain lowest energy conformation of the ligand, the overall geometry optimization was carried out by Powell method [22] using Tripos force field with a convergence criterion of 0.05 kcal/mol. Å by giving 1,000 iterations. Charge distributions were calculated by the Gasteiger-Marsili method [23].

Docking in GOLD
The GOLD software (Version 3.0) [24]

Results
The chloroform fraction was eluted from the column chromatography with hexane-acetone (65:35%) mixtures and compound 1 was isolated as a colorless powder. The IR spectrum of compound 1 showed strong absorption bands between 3409-3415 cm -1 for the hydroxyl group and at 2975 cm -1 for trans-quinolizidine moiety [25]. Hz) were assigned to the C-3 and C-6 protons, respectively [12,13].
The spectral data of compound 1 was identical to the earlier reported data of dihydroimperialine a reduced derivative of imperialine (2) [27,28]. The 13C-NMR (CDCl 3 , 100 MHz, BB and DEPT, Table   1) spectra showed signals for all 27 carbon atoms. Two downfield signals resonating at 71.9 and 73.3 were assigned to the OH-containing C-3 and C-6 carbons atoms, respectively. Two quaternary carbons at 35.5 and 70.9 were assigned to the C-10 and C-20 carbon atoms, respectively (Figure 1). (2), zygacine and eduradine [29]. The base peak at m/z 112 and 98 indicated the presence of cevanine-type steroidal alkaloids [29].
Dihydroimperialine (1)   The ring A was stabilized by hydrogen bonding between hydroxyl group A and terminal hydroxyl group of Thr120 (3.07 Å) [30]. Another much stronger hydrogen bond was observed between the hydroxyl group B and the peptidic carbonyl atom of Pro 285 (2.82 Å) (Figure 3). Additionally, the center of ring A into gorge of BChE is stabilized by cation-interaction contributed by Asp 70 . The ring B forms -interaction to one of the aromatic amino acid residues of gorge Tyr 332 . Compound 1 was evaluated for its butyrylcholinesterase inhibitory activity and found to be active against the butyrylcholinesterase enzyme with IC50 14.4±1.0 (μM). The inhibitory activity and IC 50 values are given in Table 1. Conclusion: This is the first report of the isolation of dihydroimperialine (1) form natural source and molecular docking studies showed the sign for future drug discovery for Alzheimer's disease.  (1) and BChE, showing the hydrogen bonding, cation-π and π-π interactions. Amino acid residues in numbered are dominant residues that are involved in interactions.