Protective Effects of Gossypin in Colchicine-induced Cognitive Dysfunction and Oxidative Damage in Rats

 

Jayant Singh Bhardwaj, Suman Manandhar, Sayan Chatterjee, Gangadhar Hari, Keerthi Priya, S. Raviteja, K. Sreedhara Ranganath Pai*

Department of Pharmacology, Manipal College of Pharmaceutical Sciences,

Manipal Academy of Higher Education, Manipal, 576104, Karnataka, India.

 

ABSTRACT:

Gossypin is known to have antioxidant property and its neuroprotective activity in the ischemia model has been reported. There are no reports on the activity of Gossypin in the learning and memory. The objective of the current work was to evaluate the protective effects of Gossypin in Colchicine-induced Cognitive Dysfunction in rats. For this experiment Male Wistar rats were used and were administered with intracerebroventricular injection of colchicine and oral solution of gossypin (10 or 20 mg/kg) for 3 week to determine the preventive effect of gossypin against colchicine toxicity. Exposure to colchicine produced oxidative stress in rats and model for deficit in memory. The activity of Gossypin in enhancing memory was evaluated using Morris Water Maze for parameters related to spatial memory as Escape latency, island entries, etc. followed by biochemical estimation of AChE enzyme level in the frontal cortex and Hippocampal region of rat brain. Docking interaction study for understanding affinity of Gossypin to AChE enzyme was done using Schrodinger suite. Insilico XP docking study suggested the strong affinity of Gossypin to AChE. Treatment with Gossypin showed significant reduction of the AChE level in frontal cortex but not in Hippocampal region. Gossypin treated group showed slight reduction but not significant in the latency period. However significant increase in the number of island entries was observed. Overall, Gossypin showed significant in silico interaction with AChE, increased AChE activity and slight cognition enhancing property.

 

 

 

INTRODUCTION:

The most prevalent disorder in the aging population is Alzheimer's disease (AD), which comprises 60%-70% of dementias^1,2. Almost 38.4 million people around the globe were found to be affected with AD as per studies on 2017, 7.7 million new cases of dementia are added every year³. Progressive Cognitive decline, loss of intellectual, linguistic skills, decreased functional ability and psychological symptoms are evident symptom in AD patients⁴. Neurofibrillary tangles (NFT) and Amyloid beta (Aβ) accumulation in the brain is observed in the individual with AD followed by abnormal hyperphosphorylaition of Tau protein, synaptic loss, neuronal degeneration.

 

Aβ deposition leads to mitochondrial damage and generation of reactive oxygen species, lowered ATP production and oxidative damage. ROS generation enhances release of cytokines and inflammatory markers leading to neuroinflammation and neuronal death⁵. The currently available therapies are lacking the disease modifying property and only provide the symptomatic relief. Additionally the chronic use of available medications may have severe adverse effects like cholinergic crisis, organs toxicity- hepatic, renal toxicity⁶.

 

Phytochemicals with neuroprotective property can be the safe and effective alternatives for management of dementia^7,8. Flavonoids are mainly reported to possess antioxidant, anti-inflammatory property^9,10. Gossypin also known as gossypin-8-O glucoside is a naturally occurring bioflavonoid found in Hibiscus vitifolius belonging to the family of Malvaceae. It is reported to have antioxidant, anti-inflammatory, anticonvulsant and anti-cancer activity^11,12. In this study, the neuroprotective effect of Gossypin against colchicine induced cognitive dysfunction in rats was investigated.

 

Colchicine is the well known microtubule disrupting agent that causes time and dosage dependent neurochemical deficit created by the increased free radical generation and oxidative damage¹³. Intracerebroventricular (i.c.v.) injection of Colchicine has been widely accepted model to induce sporadic dementia like Alzheimer’s due to axoplasmic flow inhibiton^14.

 

MATERIALS AND METHODS:

Chemicals and Reagent:

Chemicals and Reagents used in this study were of analytical grade. Carboxy Methyl Cellulose was purchased from SRL chemicals and Gossypin from Himedia. 5,5’-Dithiobis-(2-nitrobenzoic acid (DTNB) and Acetylthiocholine iodide were purchased from Sigma-Aldrich, USA.

 

Animals:

Male Wistar rats of 150-200g weight were procured from the Central Animal Research Facility, MAHE. Before the experiment, the animals were acclimated. After surgery, animals were maintained with standard rat pellets and drinking water ad libitum in standard condition of 12h light and dark cycle. Prior approval was obtained from IAEC, Manipal was obtained for the experimental proposal. Animal handling and other surgical procedures of the study were conducted according to CPCSEA guidelines.

 

Administration of Colchicine by Intracerebroventricular injection:

Colchicine administration was done using the Stereotaxis apparatus, with Stereodrive software preloaded with rat brain atlas. Animals were administered thiopental sodium (45mg/kg, i.p.) for anaesthesia. The animal’s head was aligned, and an midline incision was made in the scalp to expose bregma. 5μL of Artificial cerebrospinal fluid (ACSF) composition:147mM NaCl, 2.9mM KCl, 1.6mM MgCl₂, 1.7mM CaCl₂, and 2.2mM dextrose) was injected in the lateral ventricle of the brain co-ordinates (AP, -0.8mm; ML, 1.8mm; DV, -3.6mm) Colchicine 15μg in ACSF was injected using Hamilton microsyringe and was placed for 2 minutes at the injection place to avoid backflow. The scalp was sutured, and the antibiotic cream was applied. After the surgery, animals were provided with palliative care along with easy access to food and water^9,10.

 

Drugs and treatment schedule:

Animals (six rats in each group) were randomly allocated into six treatment groups as per the treatment protocol. Dementia was induced by the i.c.v. injection of Colchicine followed by continuous treatment till the end of experiment. Gossypin and Standard drug (Donepezil) suspension in carboxymethyl cellulose (CMC) and was administered orally to the groups respectively for four days prior to i.c.v. injection till the study end. The first group, normal control received only vehicle, i.e. 0.25% CMC Solution (10ml/kg, p.o.) throughout the study period. The second group, Sham control also received 0.25% CMC Solution (10ml/kg, p.o.) followed by ACSF, (5µL, i.c.v.). The animals of the remaning groups, received Colchicine+ACSF (15μg Colchicine/5μL ACSF, i.c.v.). The fourth, fifth and sixth groups were administered donepezil (2mg/kg, p.o.), Gossypin (10mg/kg, p.o.), Gossypin (20mg/kg, p.o.) respectively for 25 days. The doses of gossypin were selected as reported in the literature¹¹.

 

Spatial Memory assessment using Morris Water Maze:

Spatial memory was evaluated with Morris water maze consisting of a circular pool of 150cm diameter and height of 40cm divided into four North-east, North-west,

South-east, South-west quadrants15. The animals were placed in either of the quadrants alternating for each trial. The water in the pool was made opaque with non-fat milk and platform of 10cm diameter was placed 2cm

immersed, hidden in the water. Animal use xternal/extra-maze cues to find the platform. 16  A tracking software system with a camera was placed to capture the behavior and movements of animal. Water maintained at a temperature of 26 ± 1º C was changed every day. The experiment was performed in the dim light intensity was kept constant throughout the trial.

 

Acquisition trial:

After 16 days of colchicine administration (15μg, i.c.v.) training was provided for four consecutive days to all animals. For a period of 60 seconds animals were allowed to swim freelyin order to find the platform. If the animal does not find a platform, they are directed to the platform manually and held on it for 30 seconds. This training was conducted as four trials a day with a 5 minute intertrial interval. The path efficiency, average speed, time spent in each quadrants, escape latencies, were recorded.

 

Probe trial:

After training phase, retention trial was done on 5^th day for 60s Post acquisition trial. The platform was removed in this trial, without altering other external/extra-maze conditions. The animals were kept with head facing to the wall in the quadrant opposite to the one containing platform and were allowed to explore the pool wherein parameters like escape latency, path efficiency, average speed, time in all zones, and total distance were considered and recorded using ANY-maze software with the camera fixed above the pool.

 

Locomotor activity evaluation by actophotometer:

The locomotor activity of each animal was evaluated on days 3, 7, 14 after colchicine administration (15μg, i.c.v.), using digital actophotometer. The animals were placed on the actophotometer that contains light sensitive infrared photocells to monitor the movement of animals. The animals were acclimatized for 3 minutes and then were kept in the instrument for 10 minutes to record their locomotor activity.

 

Tissue Collection and Preparation for Biochemical estimations:

After the probe trial, animls were euthanasized using high dose of thiopental sodium. They were perfused with 20mL cold saline and two frontal cortex lobes of brain were cautiously isolated and preserved in -20°C until analysis. Before analysis, in ice cold 0.1M Phosphate buffer (pH 7.4) tissue homogenate (10% w/v) was prepared using homogenizer at 6000rpm for 15 minutes followed by the biochemical parameters measurement.

 

Estimation of Acetylcholinesterase Activity:

Acetylcholinesterase activity was estimated in hippocampus and frontal cortex¹⁷ using method described by Ellman et al, 1961^18,19. Briefly, 100µL of sample supernatant was added to 650µL of 0.1M phosphate buffer (pH7.4). To this, 5µL of 20mM acetylthiocholine iodide was added and finally 25µL of 0.5mM DTNB was added to this mixture to initiate the reaction. The absorbance was then measured at 412nm for 180 seconds.

 

Molecular docking experiment:

Docking studies were performed for identifying binding affinity of Gossypin to AChE²⁰ using Maestro suite of Schrodinger^21,22. The 2D SDF format of Gossypin molecule with molecular formula, C₂₁H₂₀O₁₃ was obtained from Pubchem and prepared for docking anaylsis by using LigPrep tool. The coversion to 3D form, generation of possible tautomers, conformers were done during Ligand Prepation. The inhibitor bound structures of Acetylcholine esterase (4M0F) with resolution of 2.304 Å was obtained from RCSB Protein Data Bank (PDB)²³. Protein preparation Wizard tool was used to prepare the protein for addition of missing hydrogen atoms, removal of unwanted water molecules and energy optimization using force field OPLS3e (Optimized potentials liquid for simulation_3e). The grid was generated based on the inhibitor bound pocket in the protein²⁴. The extra precision (XP) mode docking was performed to determine the possible interactions of Gossypin molecule with the Acetylcholinesterase receptor. The drug likeness nature of Gossypin was also evaluated using QikProp module. ADME (Absorption, Distribution, Metabolism, Excretion) property including aqueous solubility (QPlogS), Molecular weight, partition coefficient (Qlog_o/w), toxicity (QPlogHERG), % oral absorption, violation from Lipinski’s rule etc were calculated.

 

Statistical Analysis:

Graph Pad Prism software was used to analyze the results. Two Way ANNOVA followed by Tukey’s multiple compartment test was used to analyze locomotor activity. All of the other tests were analyzed using One Way ANNOVA followed by Tukey’s multiple compartment test and expressed as Mean±SEM. Experimental results after analysis were considered significant at the p value less than 0.05.

 

Figure 1: Schematic diagram for representing drug treatment plan, disease induction and evaluation of parameters.

 

RESULT:

Drug likeness properties:

The physicochemical properties of the Gossypin were predicted by using QikProp tool and tabulated in the Table1. The molecular weight of Gossypin is less than 500, good predicted CNS activity was found. Predicted IC50 value for blockage of HERG K+ channels (QPlogHERG) was observed out of range showing the possibility of toxicity. It showed score of 2 in Rule of five section depicting that Gossypin could be considered as drug-like molecule as it follows Lipinski rule of 5. The prediction for % human oral absorption is based on stastical method of multiple linear regression model correlating with the human oral absorption. However, the oral absorption percentage for Gossypin was 0 indicating its poor oral absorption.

 

Table 1: Showing the pharmacokinetic ADME predictions using QikProp tool.

Title	Mol Wt	CNS	QPlogP­o/w	QPlogS	QPPCaco	QPlogHERG	QPlogBB	Rule of 5	%Human Oral absorption
Recommended values	130.0-725.0	-2.0- 2.0	-2.0-6.5	-6.5-0.5	<25 poor, >500 great	concern below –5	-3.0-1.2	Max-4	1-low, 2-medium, 3-high
ZINC00008046	480.381	-2	-1.957	-2.439	1.156	-5.183	-4.243	2	0

 

Abbreviations: Mol Wt- Molecular weight; CNS: Predicted central nervous system activity; QPlogPo/w: Predicted octanol/water partition coefficient; QPlogS: Predicted aqueous solubility; QPlogBB: Predicted brain/blood partition coefficient, QPPCaco: Predicted apparent Caco-2 cell permeability in nm/sec; QPlogHERG: Predicted IC50 value for blockage of HERG K+ channels;

 

Molecular Docking Study:

Gosspyin showed good interaction with Acetylcholinesterase receptor with docking score of -15.508. Donepezil with docking score of -16.750, showed interaction with AChE by forming H-bond between PHE295 and carbonyl group, Pi-cation interaction with TYR341 and Pi-Pi stacking with TRP286. Similar interaction was shown by Gossypin molecule by foming H-bond interaction with PHE295, SER293, TYR72 and ASP74 residues; Pi-Pi stacking interaction with TYR341, TRP286 as shown in the Table. Gossypin showed common interactions as well as some additional interactions increasing its potential for AChE binding and afiinity. Gossypin showed better docking score of -15.508 and interactions that the inbound molecule of the PDB, Territrem which has docking score of -13.022.

 

Table 2: Showing the dock score, binding interactions of different ligands with AChE enzyme.

Ligand	2D- interaction diagram	Dock Score	Interactions
Gossypin		-15.508	H bond: ASP74, TYR72, PHE295, SER293. Pi-Pi stacking: 2TRP286, TYR341 Hydrophobic: TYR341, PHE297, PHE295, VAL294, TRP286, TYR72, LEU76, TYR124, TRP86. Polar: THR83, THR75, SER293. Charged Positive: ARG296.
Donepezil		-16.750	H-bond: PHE295 Pi-cation: TYR341 Pi-Pi stacking: TRP286 Hydrophobic: TYR341, PHE297, PHE295, Val294, TYR337, PHE338, TRP286, LEU289, TYR72, TRP86, TYR124 Polar: THR83, ASN87, SER125, SER293 Charged(positive) ARG296
Territrem B		-13.022	H-bond: TYR124, PHE295 Pi-Pi interaction: TRP286 Hydrophobic: PHE297, PHE295, VAL294, TRP286, TYR124, TYR337, PHE338, TYR341, TYR72, TRP86 Polar: HIS447, SER203, SER125, SER293, Charged(positive) GLH202, GLU292, ASP74

 

Impact of Gossypin on the locomotor activity:

The locomotor activity of rats was evaluated using Actophotometer after i.c.v. injection of Colchicine. The mean counts of locomotor activity per 10 mins were normal and stable without significant variation as shown in Figure 2. This indicates that there was no detrimental effect on locomotion due to surgery. The locomotive operation had no significant difference in all groups compared to normal and disease control.

 

Figure 2: Effect of Gossypin on locomotor activity on Day3, 7 and 14^th in cognitive impaired rats. Values are expressed as Mean ± SEM, n=6.

 

Effect of Gossypin on AChE levels of brain:

Injection of colchicine i.c.v. in the brain showed a significant rise in the level of AChE compared to the normal group and sham control group. The significant reduction in AChE activity in the frontal cortex was observed in the Donepezil and Gossypin (10 and 20mg/kg) treated groups (Figure 3A) compared to the Colchicine only treated disease group. When compared to disease control, there was no significant reduction in the hippocampal AChE activity (Figure 3B). The AChE activity in the hippocampal region was significantly elevated in the Donepezil and Gossypin treated group as compared to the normal and Sham control rats.

 

Figure 3: Effect of Gossypin on 3A) Hippocampus and 3B) Frontal cortex acetylcholinesterase (AChE). Values are expressed as Mean ± SEM, n=6. * represents p< 0.05, ** p<0.01, *** p<0.001 when compared with vehicle, and # p< 0.05, ## p<0.01 as compared to Disease control.

 

Effect of Gossypin in the spatial memory:

Escape latency refers to the time taken to find the hidden island located in D-quadrant. i.c.v. Colchichine treatment increased the escape latency compared to the vehicle control. Treatment with the standard Donepezil and Gossypin (10mg/kg) showed decreased latency period as compared to the disease group (Figure 4A). Higher dose of Gossypin (20mg/kg) did not show reduction in the Escape latency period when compared with rats of the disease group. There was no significant change in the escape latency period among all groups.

The i.c.v. Colchine treatment resulted in the less number of entries to the island region in the probe test day. However, significant increase in the number of entries was observed in Donepezil and Gossypin (10mg/kg) treated rats when compared to Disease control colchine treated group but higher dose of Gossypin had no increase in the number of entries (Figure 4C).

 

Similarly rats treated with i.c.v. Colchicine took longer time to cross D quadrant as compared to normal and Sham control rats. The significant reduction in the latency to D quadrant was observed in Donepezil treated group and Gossypin (10 and 20 mg/kg) treated groups (Figure 4B).

 

Figure 4: Effect of Gossypin on 4A) Escape Latency, 4B) D quadrant latency 4C) island entries and. Values are illustrated as Mean ± SEM, n=6.* represents p< 0.05 as compared with vehicle, # represents p< 0.05 as compared with Disease control.

 

DISCUSSION:

AD is a neurodegenerative disorder identified by reduction in cognition abilities. It is observed that colchicine injection into the brain precipitates the condition like Alzheimer’s disease⁹. Colchicine is a compound that binds to tubulin proteins resulting in dephosphorylation and destabilization. This leads to accumulation of amyloid beta(Aβ) and generates tangles similar to neurofibrillary tangles. Consequently, it triggers excessive oxidative stress, and neuroinflammation. Neuroinflammation has been considered as one of the pathological cause for AD^25,26. In the current study, Gossypin is evaluated for its neuroprotective activity against colchicine-induced cognitive dysfunction.

 

The Cholinergic hypothesis signifies the role of Acetylcholine in memory and has led to the identification and use of acetylcholinesterase inhibitors for enhancing memory and cognition^27,28. Increased AChE activity has been observed in the plasma, human liver, red blood cells, cerebrospinal fluid and brain from Alzheimer’s patients. Donepezil, a cholinesterase inhibitor was selected as a standard drug for the current study. In this study the increased level of AChE was found in the frontal cortex and Hippocamus of rat brain after i.c.v. Colchicine administration²⁹. The significant reduction in the level of AChE was seen after treatment with Gossypin (10mg/kg and 20mg/kg) as compared to the disease control rats.

 

Gossypin has been reported to have antioxidant property showing its potential to scavenge free radicals¹². Gossypin also has been observed to show dose dependent neuroprotective activity in cerebral ischemia model by its antioxidant property leading to increased Superoxide dismutase, Glutathione level and reduced lipid peroxidation level¹¹. In this study, Gossypin treatment in the i.c.v. Colchine induced cognition impared rats reversed the increased AChE level in the frontal cortex of the brain but had no significant effect in the Hippocampal region of brain.

 

Additionally, in silico docking study has provided additional support for the affininty of Gossypin to the AChE receptor. It has shown similar activity as compared to Donepezil in terms of binding docking score as well as interactions with the amino acid residues PHE295, TYR341 and TRP286. QikProp analysis for pharmacokinetic properties has predicted for less oral absorption and intestinal absorption of Gossypin which might be one of the reason for showing less potency in behavioral assays in in vivo experiments. Formulation based enhancement of bioavailability of Gossypin might be best strategy to obtain maximal potency of Gossypin in the cognition and memory enhancement.

 

The i.c.v. administration of Colchine leads to the memory impairment indicated by elevated D-quadrant latency and escape latency in Morris water maze test. Donepezil, standard drug showed reduction in the latency time as well as increased number of entries to island. Lower dose of Gossypin (10mg/kg) was capable to reduce the latency period effectively than the higher dose of 20mg/kg. However, there was no significant reduction in the latency period in both of the treatment group. This might be due to the poor bioavailability of the drug as suggested by the in silico prediction study. There was no change in the locomotory activity as observed in the results of actophotometer among the rats of different groups. This removes the possibility of the Colchicine toxicity and experimental errors affecting locomotion in the rats.

 

CONCLUSION:

In current study, neuroprotective activity of Gossypin (10 and 20mg/kg) was evaluated against i.c.v. colchicine induced cognitive dysfunction. Dose dependent activity of Gossypin could not be justified in this study. Gossypin at lower dose showed significant activity for AChE level whereas higher dose of Gossypin showed no significant effect. Based on the insilico study, Gossypin showed good interaction and binding energy with AChE enzyme in the docking study. Insilico pharmacokinetic predictions suggested low bioavailability so further study is warranted on the bioavailability and pharmacokinetic studies for Gossypin. Further formulation based study could be done to achieve good bioavailability of Gossypin.

 

ACKNOWLEDGMENTS:

The authors thank Manipal – Schrödinger Centre for Molecular Simulations and Manipal College of Pharmaceutical Sciences, Manipal Academy of Higher Education, Manipal, India for providing facility to carry out research work.

 

CONFLICT OF INTEREST:

Authors declares no potential conflict of interest.

 

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Received on 18.10.2019           Modified on 05.12.2019

Accepted on 20.01.2020         © RJPT All right reserved