Neuroprotective effect of Cubebin: A dibenzylbutyrolactone lignan on scopolamine-induced amnesia in mice

Material & Methods

This study was carried out in the department of Pharmaceutical Science and Technology, Pharmacology Laboratory, Institute of Chemical Technology (ICT), in 2012.

Scopolamine, sodium hydrogen phosphate, sodium dihydrogen phosphate and dimethyl sulphoxide (DMSO) were purchased from SD Fine Chemicals Ltd., Mumbai. DTNB [5, 5’-dithiobis-(2-nitrobenzoic acid)] reagent was purchased from Sigma, India. Acetylthiocholine iodide (ATCI) was purchased from Hi-Media, Mumbai. Adult male Wistar rats weighing 180-250 g and Swiss albino mice weighing 25-30 g were used. The experimental protocol was approved by the Institutional Animal Ethics Committee of ICT, Mumbai. The animals were maintained in standard laboratory conditions with food and water ad libitum, under a 12 h light/12 h dark cycle.

Molecular docking: The procedure of molecular docking of cubebin was followed as described elsewhere8. Cubebin structure was constructed using ‘build option’ within Maestro using standard geometries and standard bond lengths as described earlier8.

Isolation of cubebin from Piper cubeba (Kababchini) fruits: A 500 g powdered dried fruits of P. cubeba was extracted with about two litres of methanol, filtered and concentrated to obtain oily extract. Approximately 100 ml extract obtained was treated with 2g of sodium hydroxide (0.5 M NaOH) to saponify the oily matter. The dried soap obtained after saponification reaction was further extracted with 100 ml acetone 3-4 times. Acetone fraction was concentrated and dried, and subjected to silica gel column chromatography. Petroleum ether-ethyl acetate (85:15 v/v) was used as mobile phase. Of the 40 eluted fractions of 200 ml each, fraction number 19 - 30 showed presence of cubebin (2.278 g of cubebin from 500 g of crude powdered drug) which was monitored by thin layered chromatography (mobile phase- toluene: ethyl acetate at 70:30 v/v). Structure was confirmed by mass spectrometry, nuclear magnetic resonance spectrometry (done at Sophisticated Analytical Instrument Facility, IIT, Mumbai) and infra-red spectrometry (done at Dept. of Pharmaceutical Technology, ICT, Mumbai). The percentage yield of cubebin was found to be 0.23 per cent.

Experimental design: AChE enzyme was isolated from rat brain homogenate as reported earlier89. This crude enzyme was used in the study to observe inhibition by different concentrations of cubebin. The AChE activity was measured using the colorimetric method of Ellman et al10, with slight modifications utilizing acetylthiocholine iodide (ATCI) as a substrate and crude AChE from rat brain. Dose range of cubebin was selected on the basis of reported LD₅₀ value (500 mg/kg; i.p.)11; 1/10^th dose of cubebin was used for investigation. Animals were randomly divided into five groups containing six animals in each group. Group I: normal control, Group II: scopolamine (SCP) 3 mg/kg, i.p.; Group III: SCP 3 mg/kg + donepezil 1 mg/kg, Group IV: SCP 3 mg/kg + cubebin 25 mg/kg, i.p.and Group V: SCP 3 mg/kg + cubebin 50 mg/kg, i.p.

Scopolamine-induced amnesia in Morris water maze (MWW) test: This test is widely accepted for the evaluation of spatial memory and learning12. The maze consisted of a dark circular water pool (120 cm diameter×45 cm height), containing water (20°C±1°C) to a depth of 30 cm and surrounded by extra maze distal visual cues of different shapes. A circular hidden platform, 15 cm diameter, was fixed constantly in one of the quadrants and was placed 1 cm below the water surface so that the mice could escape from swimming. After two weeks of training period, the day 0 reading was recorded in which the mice were placed into the maze 180° from the platform always. The time taken by the mice to reach the platform was recorded which is called escape latency time (ELT). The actual trial was conducted 30 min after scopolamine administration. ELT was recorded with the cut-off time of 60 sec. The mice were sacrificed immediately after the water maze test and brains were isolated in ice-cold saline. The isolated brains were further homogenized in phosphate buffer at pH 7.4. The homogenates obtained were centrifuged at 850 g at 4°C for 15 min. The supernatant was used for the estimation of AChE1314, lipid peroxidation (MDA)15 and total protein16.

Statistical analysis: All results were expressed as the mean±standard deviation (SD). Statistical evaluation of the data was performed using ANOVA followed by Tukey's multiple comparisons test for significant differences using GraphPad prism software version 5; 2007 (GraphPad Software, San Diego, USA).

Results

Molecular docking of cubebin: To understand the interaction of cubebin with the catalytic site of the target enzyme, molecular docking study was undertaken. Cubebin molecule was found to bind well within the catalytic site of the enzyme showing π-π stacking with Histidine (His) 440, hydrogen bonding with Glycine (Gly)119, Gly118 and Tyrosine (Tyr) 121. This ligand also showed hydrophobic interactions with Tryptophan (Trp) 84, Phenylalanine (Phe) 330 at the choline binding site, Tyr 121 at a peripheral binding site and Phe 290 at the choline binding site. Glide score of cubebin (−10.51) indicated the stability of the cubebin enzyme complex (Fig. 1).

Fig. 1

Ligand interaction architecture of acetylcholinesterase target protein (PDB accession no. 1EVE) with the superimposed docked molecule of cubebin. ILE, Isoleucine; GLY, Glycine; TRP, Tryptophan; TYR, Tyrosine; PHE, Phenylalanine; ASP, Aspartate; HIS, Histidine; SER, Serine; ALA, Alanine.

Isolation of cubebin: The isolated compound showed colourless needle of cubebin having melting point in the range of 130-132°C. TLC indicated a single band corresponding to cubebin under ultraviolet light, which was formed with concentrated H₂ SO₄ reagent as a red-violet zone at R_f 0.56. Based on chemical and spectral analysis, the isolated compound was confirmed to be cubebin.

Inhibitory effect of cubebin on AChE: Cubebin strongly inhibited activity at 1122 μM (over 56%) and at other concentrations in a dose-dependent manner. The IC₅₀ value of cubebin was 992 μM (Fig. 2).

Fig. 2

Anticholinesterase activity of rivastigmine and cubebin (IC₅₀=992 μM) in vitro. Values are mean±standard deviation, n=3.

Effect of cubebin on scopolamine-induced impairment of learning and memory using Morris water maze (MWM) test: Effect of cubebin was evaluated on spatial memory and learning in scopolamine-induced amnesia in mice. The ELT is the time taken by the mice to locate the hidden platform in the water maze which was used as a parameter to evaluate the performance of the treated mice. The scopolamine-treated mice showed significant increase in the ELT. Moreover, administration of cubebin (25 and 50 mg/kg; i.p.) was found to decrease the ELT significantly and was comparable with standard donepezil (1 mg/kg) as shown in the Table.

Table

Effect of cubebin on scopolamine (SCP)-induced changes in escape latency (sec) using Morris water maze and oxidative stress level and acetylcholinesterase (AChE) activity of mice brain

Effect of cubebin on scopolamine-induced changes in AChE activity of the mice brain: Scopolamine (3 mg/kg, i.p.) significantly (P <0.05) increased the brain AChE activity when compared to control mice. The brain AChE activity was found to be significantly (P <0.05) decreased in cubebin (25 and 50 mg/kg) and donepezil-treated groups in comparison with the scopolamine-treated group and was comparable with the control group.

Effects of cubebin on scopolamine-induced changes of the mice brain: Scopolamine (3 mg/kg, i.p.) significantly increased the brain MDA levels compared to the control group of animals, reflecting enhanced oxidative stress. The elevated enzyme activity of brain MDA level was found to be significantly restored in the cubebin-treated group (25 and 50 mg/kg; i.p.) as compared to the scopolamine-treated group (Table).

Discussion

In this study cubebin was evaluated for inhibition of AChE activity, the latter being one of the major targets in the treatment of AD. The investigation was started by evaluating the possibility of cubebin-AChE binding through molecular modelling techniques. Cubebin was docked into the binding pocket of AChE17. The preliminary docking study showed minimum binding energies of cubebin with AChE binding pocket suggesting cubebin as a promising molecule for further exploration. Cubebin was isolated from P. cubeba and the structure was confirmed by chemical and spectral analysis. In vitro studies showed that cubebin at a concentration of 1122 μM exhibited approximately 56 per cent inhibitory activity with IC₅₀ value of 992 μM.

Based on the results obtained from initial docking studies and in vitro inhibitory assay, cubebin was further assessed in scopolamine-induced amnesia model in mice. Scopolamine, a central nervous system muscarinic antagonist, causes cognitive dysfunction leading to memory impairment which can be seen as a significant increase in the ELT in MWM test. The results suggested that cubebin decreased long-term and spatial memory impairment caused by scopolamine.

The mechanism of action of cubebin was evaluated by its effect on the levels of brain AChE ex vivo in mice with scopolamine-induced amnesia. Cubebin pre-treatment (25 and 50 mg/kg) significantly inhibited brain AChE activity. In AD patients, AChE accelerates formation of fibrils from Aβ peptides and co-localizes with Aβ in extracellular plaques. Aβ protein also regulates AChE expression in AD patient's brain18. The above results showed inhibition of AChE by cubebin and thus increased cholinergic neurotransmission.

It has been reported that scopolamine impairs short memory and acquisition of new knowledge and increases AChE activity and oxidative stress in the brain19. The drugs that reversed the scopolamine-induced amnesia might have some effects on cholinergic system, mainly on the levels of acetylcholine in brain. Oxidative stress can be due to either an increase in reactive oxygen species production or a decrease in the activity of the antioxidant enzymes such as superoxide dismutase, catalase and non-enzymatic antioxidants. These further result in increased level of MDA which indicates peroxidative damage leading to cellular degeneration. Scopolamine significantly increased the levels of MDA, a marker of cellular degeneration. Cubebin significantly restored the MDA levels similar to those observed in the normal control group. This indicates that antioxidant property of cubebin may be responsible for its neuroprotective effect against the oxidative stress induced by scopolamine, possibly by restoring the elevated enzyme activity. This model can be used to screen the molecules which specifically act through cholinergic neurotransmission since the exact link between scopolamine in the production of brain oxidative stress and cholinergic dysfunction is yet to be explored20. Although cubebin has shown weaker activity as compared to donepezil, the results obtained from this study might serve basis for lead identification and further modifications. Thus, neuroprotective role of cubebin should be further evaluated with different doses of cubebin in chronic models such as intracerebroventricular injection of streptozotocin and Aβ-induced AD in rodents21. There are a few reports which suggest that cubebin possesses dose-related clastogenic and genotoxic effects at higher doses1122. In the present study, the doses selected for cubebin were much lower than the genotoxic dose.

In conclusion, the brain AChE inhibition and antioxidant activity shown by cubebin in scopolamine-induced spatial memory impairment model in mice suggested that this phytoconstituent should be further investigated in the chronic models of AD.