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INTRODUCTION
In most countries, the numerous debilitating Neurodegenerative Disorders (NDDs) such as Alzheimer’s Diseases (AD), Huntington’s Disease (HD), Parkinson’s Disease (PD) are believed to be the main problem and impose significant economic pressure on the health care system (Amarjot et al., 2021). Neuroprotection refers to the strategies and relative mechanisms able to struggle down the Central Nervous System (CNS) against neuronal damage caused by various neuropsychiatric and neurodegenerative disorders such as Alzheimer’s disease, anxiety, cerebrovascular impairment, seizures, Parkinson’s disease, etc. (Phani et al., 2015). NDDs is a hereditary or sporadic condition that results in slow and irreversible loss of neurons and their processes (axons, dendrites, synapses) with a corresponding progressive impairment in neuronal function (Ponmalai et al., 2020). NDDs such as Alzheimer’s disease resulted in very serious problem to patients themselves and also made their society pay huge expenses (Jung et al., 2020). Parkinson’s disease is defined by the degeneration of dopaminergic neurons in the substantia nigra, which ultimately leads to the death of these neurons. The depletion of neurons leads to motor dysfunctions characterized by tremors, rigidity, and bradykinesia (Mateusz et al., 2024).
Pterocarpus marsupium Roxb. (P. marsupium Roxb) belonging to the family fabaceae is popularly known as Indian Kino tree or Bijasar or Vijaysar in Hindi (Anshul et al., 2013). The phytochemicals isolated from P. marsupium Roxb exhibit a diverse range of pharmacological activities such as antidiabetic, anti-inflammatory, antioxidant, hepatoprotective, cardioprotective, neuroprotective and wound-healing effects (Vrushali et al., 2025). Traditionally stem of P. marsupium Roxb have been used for the treatment of neurological problems (Bhupendra et al., 2012). P. marsupium Roxb has shown neuroprotective, analgesic, and memory enhancing activities in animal models (Sakshiparsutkar et al., 2025).
Oxidative stress, necrosis, cytotoxicity, ions imbalance, mitochondria dysfunction, cellular inflammation, apoptosis, increased blood-brain permeability, and morphological changes are pathological alterations in response to injuries, which aggravate medical conditions and give hints to screen alternative neuroprotection approaches (Muneeb et al., 2019).
Oxidative stress is mainly related to secondary cell death in many central nervous system disorders (Aditi et al., 2020). Living cells of man, animals and plants when exposed to external environment leads to the formation of free radicals and tissue damage resulting in diseases such as atherosclerosis, heart failure, neurodegenerative disorders, aging, cancer, diabetes mellitus, hypertension and several other diseases and are becoming increasingly recognized (Deepa et al., 2014).
Flavonoids are capable of crossing the Blood-Brain Barrier (BBB), which makes them potential agents in preventing neurodegenerative disorders (Haroon et al., 2020). Flavan-3-ols have been reported to exhibit several health beneficial effects by acting as antioxidant, anticarcinogen, cardio-preventive, antimicrobial, anti-viral, and neuro-protective agents (Patricia et al., 2008).
The aim of this study was to investigate possible neuroprotective effect of the Chloroform Fraction of Pterocarpus marsupium Roxb Bark (CFPMB) and its isolated compound (afzelechin) in Aluminium Chloride (AlCl3) induced neurodegeneration in Sprague dawley rats in order to point out the role of this plant and its isolated compound (afzelechin) as potential sources for the development of therapeutic agents for neurodegenerative disorders.
MATERIALS AND METHODS
Plant material
P. marsupium Roxb plant parts were collected from Vedhagireeswar hills, Bhavani, Erode district, Tamil Nadu state, India, during the month of November-December. It was dried under shade at temperature not exceeding 40ºC. It was authenticated by Botanical Survey of India, Coimbatore, Tamil Nadu. The authentication certificate number is BSI/SRC/5/23/2023/Tech-597 dated 17-08-2023.
Preparation of CFPMB (Harborne 1998)
About 1 kg of P. marsupium Roxb bark was dried under shade. The dried material was ground into powder by using mechanical grinder. Then this powder was passed through sieve no. 40 to get uniform powder. This bark powder was successively extracted by soxhlet apparatus with solvents of increasing polarity, i.e. n-hexane, chloroform, ethyl acetate and ethanol until the change of colour of solvent. The content of round bottom flask was evaporated in the dry air in boiling water bath to get the extract. The percentage yield, consistency and colour of the extracts were noted. Then the extracts were transferred to sterile container and labelled.
Experimental animals
Sprague Dawley rats (150-200 g) were used for the experiments. Animals were purchased from Mass Biotech (Regn No.2084/PO/RcBt/S/19/CPCSEA), Chengalpet, Tamil Nadu, India and maintained at standard housing conditions. A standard commercially available diet was provided with water ad libitum during the experiment. The animals were kept in clean and dry polycarbonate cages and maintained in a well-ventilated animal house with 12 hr light/dark cycle. All the experimental procedures were carried out in accordance with the CPCSEA guidelines. The experimental protocol was approved by Institutional Animal Ethics Committee (Approval no. JKKMMRAFCP/IAEC/2024/012, dated 20.07.2024) for conducting this study.
Acute Oral Toxicity Study (OECD 2001)
Acute toxicity study of the CFPMB and compound isolated from CFPMB (Afzelechin) were performed according to OECD-guidelines 423. Three animals of same sex were used in each group. The CFPMB and isolated compound were administered to each group at 5, 50, 300 and 2000 mg/kg (p.o) respectively. The animals were fasted over-night before the administration of extract. Animals were observed regularly for 14 days for any signs and symptoms of toxicity.
Neuroprotective effect of CFPMB and Isolated compound (Afzelechin)
In this study, CFPMB and Isolated compound (Afzelechin) were tested for their reversibility of neurodegeneration caused by AlCl3 administration at 17 mg/kg/p.o (Prachi et al., 2023) for 21 days. 6 groups of 8 rats weighing between 150-200 g in each group were divided randomly.
Group - I (Vehicle control) rats were administered with Distilled water. Group- II rats were administered with AlCl3 (17 mg/kg/p.o) only. Group - III rats were administered with AlCl3 (17 mg/kg/p.o) + Donepezil hydrochloride (3 mg/kg, p.o.) (Laxmi et al., 2017). Group IV and V were administered with AlCl3 (17 mg/kg/p.o) and CFPMB (200 mg/kg/p.o and 400 mg/kg/p.o) respectively and Group - VI rats were administered with AlCl3 (17 mg/kg/p.o) and isolated compound (30 mg/kg, p.o). After 24 hr of the administration of the last dose, animals were subjected to behavioural tests and finally sacrificed and the brain was extracted for histopathological studies and biochemical analysis.
Examination of general behaviour
Change in Body weight
The change in body weight of individual animals was recorded once in a week (Hemlata et al., 2021).
Behavioural pharmacology studies
Morris water maze test
Morris water maze test is a method to assess spatial or place learning (Ponmalai et al., 2021). It consists of an open circular pool with having a diameter of 100 cm and 50 cm in height with a featureless interior surface. A circular platform was hidden 2 cm below the water level. The circular pool was filled with water maintained at a constant temperature of 23±1ºC and camouflaged with 500 mL of milk. All the animals were trained with 3 trials per day with the interval of 5-10 min for 5 days a week. The animals were allowed to stay in the water maze for maximum period of 2 min. Each trial started from one of four assigned polar positions with a different sequence each day. The first step to calculate learning is the escape latency. It is the time taken by the animal to reach the platform.
Pole climbing test
Cook’s pole climbing test apparatus was used to study the effect of test compounds on Aluminium Chloride induced memory impaired animals. The apparatus consists of a 25 cm × 25 cm × 40 cm chamber along with dim light and sound box. The electric shock was given to animals in grid floor of the chamber. The animals avoided electric stimulus by jumping onto the pole. This jumping was noted as an escape time of the animals to avoid foot shock. However, jumping on to the pole before shock due to buzzer sound was considered as avoidance. The experiment was terminated after 5 trials with intervals of 30 sec. A significant reduction in escape latency time was considered as successful retention of avoidance memory (Shukla et al., 2021).
Elevated Plus Maze Test (EPM)
EPM is a conflict paradigm that consists of two closed arms (length 30 cm × width 5 cm × height 15 cm), two open arms (length 30 cm × width 5 cm), and a central platform (5 cm × 5 cm). The maze was elevated 15 cm above in a dimly illuminated room. Rats were placed individually into the center of the maze, facing open arms. The time spent and number of entries in open arms was recorded for 5 min. Increased activity in the open arms was interpreted as an index of potential anxiolytic activity (Kumar et al., 2021).
Open Field Test (OFT)
The OFT provides a useful method to simultaneously measure of the exploratory behaviour, total locomotor activity levels and anxiety-like behaviour in rodents. The test is based on the tendency of the rodents to explore the novel area and avoid bright light. The open field is an enclosed space, with surrounding walls that prevent the animal from escaping. The field is marked with a grid and square crossings. The center of the field is marked with a different colour to differentiate from the other squares. On the 22nd day of the experiment, the animals were subjected to OFT. The animals were placed in the open field and observed for 5 min. The behaviour assessments included line crossings referring the rate with which the animals cross a grid line with all four paws. These behaviours indicated change in exploratory behaviour (Kada et al., 2022).
Estimation of Biochemical parameters
Brain Tissue Homogenate Preparation
Rats were sacrificed with mild ether anaesthesia and brain dissected out, washes it thoroughly with saline solution, and divided into two halves. One-half of the brain of each rat was homogenized instantaneously in a solution containing Tris-HCl (50 mM, pH 7.4) and sucrose (300 mM). The tissue homogenate was centrifuged at 10000 RPM for 10 min at 4ºC, and the supernatant was separated for the biochemical estimation (Ramachandran et al., 2020).
Estimation of Acetylcholine (Ach) content
1 mL of rat brain homogenate was placed in a boiling water bath for 5 min to terminate the Acetylcholinesterase enzyme activity and also to release the bound ACh. To the homogenate, 1 mL of alkaline hydroxylamine hydrochloride was added followed by 1 mL of 50% hydrochloric acid solution. The contents were mixed thoroughly and centrifuged. To the supernatant, 0.5 mL of0.37 M ferric chloride solution was added and the brown colour developed was read at 540 nm against a reagent blank (1 mL of alkaline hydroxylamine hydrochloride +1 mL of 50% hydrochloride + 1 mL of distilled water + 0.5 mL of 0.37 M ferric chloride solution) in a spectrophotometer. The Acetylcholine content was expressed as μ moles of ACh/gm wet weight of tissue (Santhi et al., 2020).
Determination of AChE Activity
The brain concentration of acetylcholinesterase was estimated by the method of Ellman et al with slight modification. Brain homogenate (0.1 mL) was mixed with 6 mL of 0.1M sodium phosphate buffer (pH 8), 0.2 mL of 0.075M acetylthiocholine iodide, and 0.01 mL of 0.01M5,5’-dithio-bis- (2-nitrobenzoic acid (DTNB, the Ellman reagent). The changes in the absorbance of the mixture were measured at 412 nm. The results were expressed as μM of acetylthiocholine hydrolysis per milligram of protein (Ponmalai et al., 2021).
Estimation of Dopamine level
The dopamine level in rat brain was estimated by admixing of homogenised supernatant liquid (1 ml) with 1 mL of ferric chloride (1.5 × 10-2 M) and 1 mL of potassium ferricyanide (1.5 × 10-2 M) in 25 mL distilled water. It was kept aside for 30 min and the developed colour was estimated using the UV-visible double beam spectrophotometer at 735 nm. The results were expressed as mole of dopamine per mL of supernatant liquid (Chandravadivelu et al., 2019).
Estimation of Glutamte content
The supernatant from brain homogenate was evaporated to dryness at 70ºC in an oven and the residue was reconstituted in 100 mL double distilled water. The 2 mM glutamate standard solution with the sample was spotted on Whatman no.1 chromatography paper using a micropipette. It will be placed on a chamber containing butanol: acetic acid: water (12: 3: 5 v/v) as solvent. When the solvent front reached the top of the paper, it was removed and dried and sprayed with ninhydrin reagent and placed in an oven at 100ºC for 4 min. The portions which carry glutamate corresponding with the standard was cut and eluted with 0.005% CuSo4 in 75% ethanol. Their absorbance was read against blank at 515 nm in spectrophotometer. The concentration of glutamate was expressed as µmol/gram wet weight tissue (Phani et al., 2017).
Evaluation of antioxidant activity
Estimation of Superoxide Dismutase (SOD)
The supernatant (500 μL) was added to 0.8 mL of carbonate buffer (100 mM, pH 10.2) and 100 μL of epinephrine (3 mM). The change in absorbance of each sample was then recorded at 480 nm in spectrophotometer for 2 min at an interval of 15 sec. Parallel blank was run for determination of SOD activity. One unit of SOD is defined as the amount of enzyme required to produce 50% inhibition of epinephrine auto-oxidation. The reaction mixtures are diluted 1/10 just before taking the readings in the spectrophotometer (Prakash et al., 2017).
Estimation of Catalase (CAT)
The activity of CAT was measured as the amount of hydrogen peroxide consumed per minute per milligram of the protein assayed by the method of Takahara et al. (1960). To 1.2 mL of 50 mM phosphate buffer pH 7.0, 0.2 mL of the tissue homogenate was added and reaction was started by the addition of 1.0 mL of 30 mM H2O2 solution. The decrease in absorbance was measured at 240 nm at 30 sec intervals for 3 min. The enzyme blank was run simultaneously with 1.0 mL of distilled water instead of hydrogen peroxide. The enzyme activity was expressed as nanomoles of H2O2 decomposed per minute per milligram protein (Manju et al., 2010).
Determination of Nitric oxide (NO)
The production of NO in the brain may occur due to oxidative stress and it can be determined by estimation of nitrite level. The nitrite level was determined spectrophotometrically with Griess reagent (0.1% N-1-naphthyl ethylene amine dihydrochloride, 1% sulphanilamide and 2.5% phosphoric acid). Brain homogenate and Griess reagent was mixed equally and this mixture was incubated for 10 min and the absorbance was measured at 546 nm. The standard curve of sodium nitrite was prepared and the concentration of nitrite in the supernatant was determined from standard curve (Souravh et al., 2015).
Estimation of Protein
Protein concentrations of the tissue homogenates were determined by the standard method of (Lowry et al., 1951) using bovine serum albumin as the standard. As per this method, colour change of the sample solution was in proportion to protein concentration which was measured using colorimetric techniques. 1 mL of brain homogenate was taken and mixed with 10 mL buffer (N/10 Acetic acid and N/10 Sodium acetate). Then it was centrifuged at 2500 rpm and supernatant was collected. About 0.5 mL supernatant was taken and 0.5 mL distilled water was added to it, and to this about 5 mL of alkaline solution (NaOH+ Sodium potassium tartarate) and 0.5 mL of folin reagent was added. The optical density was measured at 600 nm (Charushila et al., 2024).
Estimation of brain Lipid Peroxidation
The quantitative measurement of lipid peroxidation in the brain was performed according to the method of Wills (1966). In this, 0.1 mL of supernatant was incubated with 0.5 mL Tris-HCl (0.1 M, pH 7.4) for 2 hr. To this, 1 mL of trichloroacetic acid (10%, w/v) was added and centrifuged at 1,000×g for 10 min. To 1 mL supernatant, 1 mL (0.67%, w/v) Thiobarbituric Acid (TBA) was added and kept in the boiling water bath for 10 min, cooled, and added 1 mL distilled water. The amount of lipid peroxidation products was measured by reaction with TBA at 532 nm using the spectrophotometer (UV-1700, Shimadzu, Japan). The values were calculated using molar extinction coefficient of chromophore (1.56×105 M−1 cm−1) and expressed as micromoles per milligram protein (Dinesh et al., 2012).
Histopathological Studies
After behavioural studies, one rat from each group was sacrificed under mild ether anaesthesia and brain were immediately removed and preserved in 10% formalin and send for histopathological examination (Srinivasa et al., 2015).
Statistical Analysis
The experimental results were reported as means with SEM. Statistical analysis was performed using GraphPad Prism software. Analysis of variance (ANOVA) and Tukey’s Multiple Comparison test were used to compare the experimental groups with the controls. p ˂ 0.05 were considered as statistically significant.
RESULTS
Acute toxicity of CFPMB and its isolated compound (Afzelechin)
There was no mortality and the animals displayed normal behaviour and did not reveal any abnormality or pathological significance. We found that the CFPMB and its isolated compound (afzelechin) were safe when given orally and no drug-related toxicity was observed even at the highest dose 2000 mg/kg and 300 mg/kg respectively.
Effect of CFPMB and Afzelecin on body weight
In comparison to the Normal control group animals, the AlCl3 only treated animals were found to be decreased in body weight throughout the 21-day treatment period. Treatment with CFPMB (200 mg/kg, 400 mg/kg) and isolated compound (30 mg/kg) prevented the decrease in body weight due to AlCl3 treatment (p<0.001) as shown in Table 1.
| Group (n=6) | Treatment | Day 0 (g) | 7th Day (g) | 14th Day (g) | 21th Day (g) |
|---|---|---|---|---|---|
| I | Distilled water (p.o) | 184.67±1.33 | 187.66±1.892 | 188.67±2.17 | 190.67±2.17 |
| II | AlCl3 (17 mg/kg/p.o) | 182±1.16 | 175.67±1.59* | 165.66±1.89*** | 160.66±1.89*** |
| III | AlCl3 (17 mg/kg/p.o)+ Donepezil hydrochloride (3 mg/kg, p.o.) | 185.67±1.59 | 187.66±1.892ns | 190.67±1.52*** | 192.67±1.52*** |
| IV | AlCl3 (17 mg/kg/p.o)+ CFPMB (200 mg/kg, p.o) | 184±1.71 | 184.67±1.33ns | 185.67±2.17*** | 186.67±2.17*** |
| V | AlCl3 (17 mg/kg/p.o) + CFPMB (400 mg/kg, p.o) | 185.67±1.59 | 188.67±2.17* | 190.67±1.52*** | 192.67±1.52*** |
| VI | AlCl3 (17 mg/kg/p.o)+ Afzelechin (30 mg/kg, p.o) | 185.67±1.59 | 187.67±1.59*** | 189.67±1.59*** | 191.67±1.59*** |
Effect of CFPMB and its isolated compound (Afzelechin) on Behavioural parameters
Administration of AlCl3 (17 mg/kg/p.o) produced significant impairment in learning and memory in AlCl3 only treated group when comparing to the normal control group (p<0.001) (Table 2). This impairment was indicated by increase in escape latencies in water maze test to reach the hidden platform and increase in Escape latency (sec) in Pole climbing test to jump to the pole to avoid electric shock and decrease of time spent in open arm and increase of time spent in closed arm in Elevated plus maze test and decreased number of lines crossed in open field test.
| Group (n=6) | Treatment | Escape latency (sec) in water maze test | Escape latency time (sec) in Pole climbing test | Time spent in Arms (sec) in Elevated plus maze test | Number of line crossed in Open Field Test | |
|---|---|---|---|---|---|---|
| Open Arm | Closed Arm | |||||
| I | Distilled water (p.o) | 15.00 ± 1.18 | 8.83±0.65 | 49.00±7.47 | 249.17±7.84 | 70.00±10.36 |
| II | AlCl3 (17 mg/kg/i.p) | 100.00± 6.41** | 83.33±3.89*** | 13.83±2.12*** | 286.17±2.12*** | 56.00±8.08ns |
| III | AlCl3 (17 mg/kg/p.o) + Donepezil hydrochloride (3 mg/kg, p.o) | 16.83±1.64** | 25.17±0.95*** | 40.67±4.00** | 259.33±4.00** | 74.33±10.92 ns |
| IV | AlCl3 (17 mg/kg/p.o) + CFPMB (200 mg/kg p.o) | 45.67± 1.94** | 84.50±1.65ns | 19.17±6.18ns | 279.17±6.36 ns | 45.50±11.74 ns |
| V | AlCl3 (17 mg/kg/p.o) + CFPMB (400 mg/kg p.o) | 34.33±2.06** | 37.50±0.76*** | 30.17±1.54 ns | 269.00±1.73 ns | 70.67±5.63 ns |
| VI | AlCl3 (17 mg/kg/p.o) + Afzelechin (30 mg/kg, p.o) | 17.17±1.99** | 32.33±4.22*** | 38.00±2.02** | 261.33±2.16** | 72.83±7.68 ns |
Effect of CFPMB and its isolated compound (Afzelechin) on Biochemical parameters
Administration of AlCl3 (17 mg/kg/p.o) produced significant decrease in acetylcholine and dopamine level and increase in acetylcholinesterase and glutamate level in AlCl3 only treated group when comparing to the normal control group (p<0.001) (Table 3). The rats that treated with CFPMB (400mg/kg /p.o) and Afzelechin (30 mg/kg/p.o) exhibited increase in acetylcholine and dopamine level and decrease in acetylcholinesterase and glutamate level when compared to AlCl3 only treated group (p<0.001) (Table 3).
| Group (n=6) | Treatment | Acetylcholine level (μ moles of Ach/gm wt of wet tissue) | Acetylcholinestrase (AChE) activity (μM of acetylthiocholine hydrolysis per milligram of protein) | Dopamine level (Mole/mL)×10−5 | Glutamate level (µ moles/gram wet weight tissue) |
|---|---|---|---|---|---|
| I | Distilled water (p.o) | 0.959 ± 0.039 | 87.75±2.48 | 2.27±0.09 | 1.41±0.04 |
| II | AlCl3 (17 mg/kg/i.p) | 0.237±0.006*** | 223.42 ± 3.53*** | 0.46 ± 0.03*** | 3.68±0.19*** |
| III | AlCl3 (17 mg/kg/p.o) + Donepezil hydrochloride (3 mg/kg, p.o.) | 0.828 ± 0.026*** | 101.46 ± 2.13*** | 2.16 ± 0.02*** | 1.65±0.06*** |
| IV | AlCl3 (17 mg/kg/p.o) + CFPMB (200 mg/kg, p.o) | 0.465 ± 0.025** | 180.78 ± 3.28*** | 1.04 ± 0.02*** | 3.03±0.15* |
| V | AlCl3 (17 mg/kg/p.o) + CFPMB (400 mg/kg, p.o) | 0.617 ± 0.031*** | 141.24 ± 2.17*** | 1.66 ± 0.09*** | 2.72±0.06*** |
| VI | AlCl3 (17 mg/kg/p.o) + Afzelechin (30 mg/kg, p.o ) | 0.776 ± 0.046*** | 121.19 ± 1.66*** | 2.00 ± 0.03*** | 2.07±0.07*** |
Effect of CFPMB and its isolated compound Afzelechin on antioxidant system
AlCl3 (17 mg/kg/p.o) only treated group showed significant decrease in Super oxide dismutase, Catalase and Total protein level and increase in Nitric oxide and TBARS level when comparing to the normal control group (p<0.001) (Table 4). The rats that treated with CFPMB (400 mg/kg/p.o) and Afzelechin (30 mg/kg/p.o) exhibited increase in Super oxide dismutase, Catalase and Total protein level and decrease in Nitric oxide and TBARS level when compared to AlCl3only treated group (p<0.001) (Table 4).
| Group (n=6) | Treatment | Superoxide Dismutase (SOD) level (units/min/mg protein) | Catalase level µmoles of catalase/mg protein | Nitric Oxide (NO) level (μmole/ mL of tissue) | Total protein (mg/mL) | TBARS (μmol/mg protein) |
|---|---|---|---|---|---|---|
| I | Distilled water (p.o) | 1.87±0.03 | 377.17±16.00 | 91.17 ± 2.79 | 1.74±0.07 | 3.73±0.22 |
| II | AlCl3 (17 mg/kg/i.p) | 0.35±0.14 ns | 43.19±5.16*** | 452.33±18.15*** | 0.40 ± 0.06*** | 8.41 ± 0.20*** |
| III | AlCl3 (17 mg/kg/p.o) + Donepezil hydrochloride (3 mg/kg/p.o) | 1.81±0.03 ns | 364.42±21.50*** | 115.33 ± 6.55*** | 1.61 ± 0.05*** | 4.02 ± 0.05*** |
| IV | AlCl3 (17 mg/kg/p.o) + CFPMB (200 mg/kg/p.o) | 0.93±0.02ns | 169.47±22.71*** | 310.33 ± 1.38*** | 0.81 ± 0.06** | 6.66 ± 0.12*** |
| V | AlCl3 (17 mg/kg/p.o) + CFPMB (400 mg/kg/p.o) | 1.39±0.04 ns | 285.42±8.06*** | 240.50 ± 1.43*** | 1.02 ± 0.05*** | 5.50 ± 0.15*** |
| VI | AlCl3 (17 mg/kg/p.o) + Afzelechin (30 mg/kg/p.o ) | 1.74±0.04 ns | 326.33±16.25*** | 159.50 ± 12.68*** | 1.33 ± 0.04*** | 4.63 ± 0.05*** |
Histopathological Studies
Effect of CFPMB and its isolated compound Afzelechin on Cerebral cortex and Hippocampus
The rats treated with Afzelechin (30 mg/kg/p.o) exhibited best regenerative and healing property and CFPMB (400 mg/kg /p.o) treated rats exhibited good regenerative and healing property in par with standard drug donepezil treated group. CFPMB (200 mg/kg /p.o) treated rats exhibited least regenerative and healing property.
DISCUSSION
Many age-related problems and neurodegenerative diseases are driven by enhanced oxidative stress, therefore the search for natural medicinal agents that boost cognitive function and enable neuroprotective effects via antioxidant action is of significant interest (Danni et al., 2023). In the current study, CFPMB and its isolated compound (Afzelechin) alleviated the behavioural, biochemical, and neurochemical problems and have potential as a neuroprotective drug.
Administration of AlCl3 (17 mg/kg/p.o) produced significant impairment in learning and memory in AlCl3 only treated group when comparing to the normal control group (p<0.001) (Table 1). This indicates that there is a severe degeneration in the brain as a result of exposure to AlCl3, which contributes to deficits in episodic memory and recognition capacity. Aluminium is a pro-oxidant and indirectly results in the production of free radicals leading to oxidative damage and reduced levels of ROS, which indirectly affect acetyl cholinesterase enzyme activity (Suvarchala et al., 2020). The possible mechanism of AlCl3 induced neurotoxicity may involve severe oxidative stress followed by inflammatory changes leading to neurodegeneration. AlCl3 can cause degeneration of cholinergic nerve terminals in cortical and hippocampus areas leading to cellular depletion and severe learning disability (Mundugaru et al., 2017).
The Morris maze was employed to assess spatial memory (Venkataramaiah et al., 2018) and animals administered with AlCl3 alone had taken longer time to reach the position of the hidden platform. Treatment with CFPMB and Afzelechin decreased the escape latency in morris water maze test. Cooks pole climbing apparatus is widely used to assess the ability of an animal to acquire, retention and retrieve the memory process and which was indicated by increasing number of avoidance response (Reddy et al., 2020). Treatment with CFPMB and Afzelechin decreased the escape latency time in Cooks pole climbing test. The Elevated Plus Maze task is widely used to predict the anxiety responses of drugs in rodents (Sahba et al., 2019). Animals which were received only AlCl3 decreased in the number of entries in open arm, and increased in the number of entries in closed arm. The treatment with CFPMB and Afzelechin reverse effects on AlCl3 induced neurodegeneration in rats. The open field test is a criterion for detecting the level of neuronal excitability and analysing the influence of drug therapy on general behavior (Gagarani et al., 2022). The memory deficit caused by AlCl3 was reversed in animals treated with CFPMB and Afzelechin demonstrates the elevated memorizing capacity of CFPMB and Afzelechin. Improvement in behavioural abnormalities may due to the presence of flavonoids in CFPMB as flavonoids have been reported to have neuroprotective effects (Paolo et al., 2023).
The cholinergic function is vital and is actively involved in the process of learning and memory, and its alteration directly leads to the development of cognitive impairment (Amandeep et al., 2021). The dopaminergic system plays important roles in neuromodulation, such as motor control, motivation, reward, cognitive function, maternal, and reproductive behaviours (Marianne et al., 2018). AlCl3 only Administered rats showed decrease in acetylcholine and dopamine level (p<0.001) (Table 3) and Treatment with CFPMB and Afzelechin reversed acetylcholine and dopamine levels.
Acetylcholinesterase (AchE) is a broadly distributed particularly potent enzyme in the brain that has several roles in cholinergic and neuromuscular synapses and is linked with building and preserving learning memory in the brain (Danni et al., 2023). The elevations in AChE are either a direct result of the neurotoxic effect of metals or due to increased lipid peroxidation. AchE is a biologically important enzyme that hydrolyses acetylcholine thereby terminating the cholinergic neurotransmission (Pandy et al., 2022). AChE inhibition is an important target for the management of Alzheimer disease (Natalie et al., 2015). Glutamate is a neurotransmitter that can cause excitatory neurotoxicity when its extracellular concentration is too high, leading to disrupted calcium balance and increased production of reactive oxygen species (Huizhen et al., 2024). AlCl3 only Administered rats showed increase in acetylcholinesterase and glutamate level (p<0.001) and Treatment with CFPMB and Afzelechin reversed acetylcholinesterase and glutamate level (p<0.001). The active constituents like phenolics, flavonoids, had shown improvement in cognitive function by inhibiting AChE (Suvarchala et al., 2020).
AlCl3 only treated rats shown elevated oxidative stress, as evidenced by a significant rise in lipid peroxidation as well as nitrite concentrations, along with a reduction in superoxide dismutase, catalase, and total protein levels in our study. The rats treated with CFPMB (400mg/kg /p.o) and Afzelechin (30 mg/kg/p.o) exhibited increase in Catalase and Total protein level and decrease in Nitric oxide and TBARS level (p<0.001) (Table 4). Neuroprotective activity of CFPMB and its isolated compound Afzelechin is related to its antioxidant properties, due to which the susceptible neurons are subjected to less oxidative stress leading to reduced neuronal harm and enhanced neuronal function.
The hippocampus and Dentate Gyrus of brain are mainly responsible for memory formation (Bindhu et al., 2019). Hippocampus and cortex neuronal are normal and healthy in normal control group rats (Figure 1) whereas hippocampus and cortex neuronal cell shrinkage was observed in AlCl3 (17 mg/kg/p.o) only treated rats (Figure 2). Treatment with Donepezil hydrochloride (3 mg/kg, p.o.) protected hippo¬campus and Dentate Gyrus neurons from AlCl3-induced neurotoxicity (Figure 3). Treatment with CFPMB (200 mg/kg p.o) had some effect on hippocampus and dentate gyrus neuronal damage caused by AlCl3 (17 mg/kg p.o.) but the results were not satisfactory (Figure 4). Treatment with CFPMB (400 mg/kg /p.o) (Figure 5) and Afzelechin (30 mg/kg/p.o) (Figure 6) protected hippocampus and Dentate Gyrus neurons from AlCl3 induced neurotoxicity. Treatment with CFPMB and Afzelechin provided a good intracellular antioxidant potent agent against oxidative stress variables. The good neuroprotective action of CFPMB and Afzelechin was exhibited in best regenerative and healing property in histopathological studies.
CONCLUSION
In conclusion, the present study reveals that CFPMB (400 mg/kg /p.o) and Afzelechin (30 mg/kg/p.o) effectively ameliorates AlCl3 induced motor deficits, cognitive impairment, and oxidative stress through its various neuroprotective mechanisms. The current study indicates that the underlying mechanism of CFPMB and Afzelechin may involve modulations of the cholinergic system, dopamine and glutamate pathway and the reduction of oxidative stress. The active principles such as flavonoids and poly phenolic compounds present in the CFPMB may be active against neurodegenerative disorders such as Alzheimer and parkinsonism. The findings of the current study propose that CFPMB and Afzelechin (flavon-3-ol) isolated from CFPMB may be a possible therapeutic option for neurodegenerative disorders.