Neuroprotective Activity of Gliricidia Sepium Leaf Extract Against Beta-Amyloid Induced Neurotoxicity in SH-SY5Y Cell Line

Neurodegenerative disorders (NDs) belong to a broad category of conditions characterised by progressive death of neurons, leading to movement dysfunction, hippocampal atrophy, cognitive decline, neuroinflammation, metabolic issues and other neurological symptoms. The neurodegenerative diseases encompass a broad array of conditions, such as Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), Huntington’s disease (HD), along with several other disorders^[1]. Neurodegeneration is acknowledged as the key pathophysiological alteration in most of the brain-related conditions^[2]. In such a scenario, neuroprotection contributes significantly. Neuroprotection provides preservation, recovery, or regeneration of the cells, structure, and function of the nervous system^[3]. As per WHO reports, approximately 3 billion individuals globally - about one in three suffer from various types of neurological disorders. By the year 2050, it is projected that there will be around 139 million dementia cases globally, with the occurrence of neurodegenerative diseases anticipated to rise even more^[4]. Alzheimer's disease is the most prevalent of all the neurodegenerative diseases, affecting over 55 million individuals globally^[5].

ALZHEIMER’S DISEASE

Alzheimer’s disease (AD) is a complex condition influenced by multiple genes and factors, marked by the accumulation of beta-amyloid peptides and hyperphosphorylated tau protein in the brain, causing plaques and neurofibrillary tangles (NFTs), which ultimately lead to dendritic dysfunction, neuronal death, memory impairment, behavioural alterations, and organ failure^[6].

Aetiology of Alzheimer’s disease

In addition to the crucial involvement of Aβ and tau, various other elements may play a role in the pathology of AD, including acetylcholine scarcity, neuroinflammation, oxidative damage, bio-metal imbalances, glutamate disruption, insulin insensitivity, gut microbiome irregularities, cholesterol homeostasis issues, Down syndrome, mitochondrial impairment, and autophagy dysfunctions^[7]. Acetylcholine is a key neurotransmitter that influences cognitive function across different regions of the brain. Genetic factors have been linked to both early and late onset AD. Mutations in APP, PSEN1 or PSEN2 lead to overproduction of pathological Aβ fragments and consequently to amyloid pathology with increased plaque formation. Alterations to chromosomes 1, 14, and 21 are associated with early-onset AD, whereas the presence of APOE- ε4 allele a form of the apolipoprotein E gene on chromosome 19 increases risk of developing late-onset AD.^[8]

Pathogenesis of Alzheimer’s disease

Two pathological features of AD are present, including senile amyloid plaques that contain the amyloid-beta (Aβ) peptide, and neurofibrillary tangles formed by hyperphosphorylated tau protein. A further crucial discovery is brain atrophy, particularly in the hippocampus^[9]. The Aβ hypothesis suggests that Aβ accumulation is critical to AD, leading to Aβ plaques, cell death, and dementia. Aβ peptide results from the improper cleavage of a larger Amyloid precursor protein (APP). APP is a membrane-spanning protein that is typically cut by enzymes known as secretases to generate different peptide fragments vital for neuronal activities. Long species (Aβ42) have a higher tendency to aggregate compared to short species. Aβ oligomers, being smaller clusters of Aβ peptides, are regarded as especially harmful to neurons and synapses. In addition to the accumulation of amyloid-beta plaques, another hallmark pathology of AD is the presence of neurofibrillary tangles (NFTs) composed of abnormally hyperphosphorylated aggregates of tau protein^[10]. Phosphorylated Tau aggregates to form oligomers that later mature into paired helical and straight filaments, which are the major components of neurofibrillary tangles^[11]. The formation and accumulation of NFTs disrupt neuronal function and contribute to neurodegeneration.  

Clinical manifestations

AD is progressive and develops over the years, resulting in the gradual emergence of cognitive impairments^[12]. It is marked by early memory difficulties and mental deterioration that may eventually impact behaviour, language, visual-spatial awareness, along with the presence of the following impairments: aphasia, apraxia, agnosia, or cognitive, behavioural and emotional problems and the motor system, and it is the most prevalent type of dementia^[13]. These conditions lead to difficulties in daily activities and indicate a cognitive and functional deterioration^[12]. Along with these symptoms, like psychotic features, aggression, depression, sleep disturbances, and apathy are also significant^[12].

Complications of Alzheimer’s disease

Complications that correlate to AD can be broadly put into 2 categories: mental/behavioural and physical complications. Mental/behavioural complications include depression, difficulty concentrating, social withdrawal, mood changes, sleep disturbances, wandering, agitation, delirium, and sundowning – common in more advanced stages^[14].  Physical complications include difficulty swallowing and breathing, aspiration pneumonia, loss of appetite, incontinence and urinary tract infection^[15].

Management of Alzheimer’s disease

Alzheimer's disease presents one of the most significant challenges in medical care this century and is the leading cause of dementia, yet it remains incurable. A multifactorial approach, involving both pharmacological and non-pharmacological intervention, is required for the management of Alzheimer’s disease^[16].  Pharmacological intervention involves the use of various categories of drugs for the treatment and management of AD and its related symptoms. Acetylcholinesterase inhibitors (AChEIs) and N-methyl-d-aspartate receptor (NMDAR) antagonists are the FDA-approved classes of drugs for the treatment of AD^[17]. AChEIs inhibit the action of acetylcholinesterase and maintain the ACh levels at the synaptic cleft. Three medications have been approved by the FDA in this category: donepezil, galantamine, and rivastigmine. These agents prevent the degradation of acetylcholine by binding to the cholinesterase enzyme. NMDA receptor antagonists drug, Memantine works by inhibiting NMDA receptors, which are voltage-dependent cation channels, thus stopping their excessive activation and lowering calcium entry into the cells, since calcium triggers excitotoxicity that results in neuronal death. It is recommended for the treatment of moderate to severe AD^[8]. It helps in memory and learning, neuroplasticity, and neuronal control.  Nootropics are a class of agents that are widely used in the management of Alzheimer’s disease. Also known as smart drugs, brain boosters or memory-enhancing agents, nootropics are substances that can enhance mental abilities such as memory, attention and concentration^[21]. Nootropics can be classified as synthetic agents, vasodilators or cerebral metabolism enhancers, and natural compounds^[22]. Dietary supplements that include vitamins, nutraceuticals and other plant-derived bioactive constituents are gaining wide popularity in the management of Alzheimer’s disease. Vitamin E is an antioxidant that is prescribed as an adjuvant therapy for AD patients^[23].

Newer Therapeutic Interventions

Although conventional medications like acetylcholinesterase inhibitors and NMDA receptor blockers are accessible for treating AD-related dementia, limited drug penetration through the blood-brain barrier (BBB) and disease relapse from ageing continue to pose difficulties^[6]. Many advanced and site-specific therapies have evolved for the treatment of AD. Monoclonal antibodies (MABs) that target amyloid have marked the beginning of a new age in therapeutics for Alzheimer's disease with targeted therapy. Three agents—aducanumab, lecanemab, and donanemab have received FDA approval, while additional ones are undergoing development. These agents are given intravenously, and upon reaching the brain, they stimulate microglia to absorb amyloid-beta protein fibrillar plaques^[24]. Genetic therapies, in general, refer to any techniques that alter genes or their expression, while gene therapies specifically focus on the genes themselves. RNA-based or gene silencing treatments can lower the synthesis of proteins like amyloid or tau by modifying the amount of mRNA that serves as the template for protein translation^[25].  Owing to an increase in neurodegenerative diseases, the challenge of delivering and releasing drugs into the brain is attracting much attention. Soft nanoparticles, such as liposomes and exosomes, are nanovesicles that have the potential to deliver drugs and genes across the BBB. The role of apolipoprotein E (ApoE) as a pivotal factor related to Aβ level and amyloid deposition in the progression of AD. Liposomes can be used to deliver plasmid-encoded ApoE2 (pApoE2) for the effective treatment of AD. The exosomes contain non-coding RNAs, microRNAs, mRNAs, lipids, and proteins. There have been successful attempts where the exosomes loaded with siRNA are used for effective brain delivery. ^[26] Stem cell therapy is another area of intensive research aiming at the treatment of Alzheimer’s disease. Stem cell therapies, including human embryonic stem cells or neural cells derived from induced pluripotent stem cells^[27].

Importance of Herbal Medicines in Alzheimer’s Diseases

The rising interest in herbal remedies stems from the therapeutic capabilities and enduring advantages of phytochemicals. Numerous traditional plants are utilized to treat cognitive and neurodegenerative conditions involved in Alzheimer’s disease^[28]. Herbal medications contain bioactive substances like flavonoids, alkaloids, glycosides, terpenes, and tannins, which are usually regarded as effective with few side effects^[29]. In traditional Indian medicine, various plants such as Allium sativum, Bacopa monniera, Centella asiatica, Withania somnifera, Ginkgo biloba, Curcuma longa, Celastrus paniculatus, Terminalia chebula, and Crocus sativus, among others, exhibit promising potential against Alzheimer's disease^[30].

MATERIALS AND METHODS

Plant Profile

Gliricidia sepium (Jacq.) Kunth. ex. Walp is a medium-sized, semi-deciduous tree that typically grows in the tropical and subtropical areas^[31]. Belonging to the family of Fabaceae, the plant is widely available in the Western Ghats of India, and studies have reported with mainly anti-inflammatory, antioxidant, anthelmintic, antimicrobial and wound healing properties. Fig.     No.1 shows the leaves of Gliricidia sepium used in the present study.

Fig. No.1 Gliricidia sepium leaf

Various parts of Gliricidia sepium  have several ethnomedical uses. The leaves of the plant are used to prepare teas to treat cough and asthma. A decoction of the leaf is utilized to cure urticaria, rash, and burns. The whole plant is used to treat headache, fever, burns, bruises, ulcers, wounds, and rheumatism, as an expectorant and sedative^[32]. The phytochemical examination of the plant revealed numerous bioactive phytoconstituents, with key components being flavonoids such as isoquercetin and isorhamnetin, along with isoflavans, 3-hydroxy-9-methoxy pterocarpan, kaempferol -O-glycosides, triterpene saponins like gliricidoside A and B, and coumarin^[33]. Each of these agents exhibits notable neuroprotective properties.

Chemicals and Reagents

Ethanol (95%), SH-SY5Y Human neuroblastoma cell line (NCCS Pune), Dulbecco’s Modified Eagle's Medium (DMEM) (Sigma Aldrich, USA D5648), β-amyloid_45-55 (10µM), 0.25% Trypsin (Invitrogen, USA 25200-056), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma Aldrich M5655), Foetal Bovine Serum (Gibco, US origin), Phosphate-buffered saline (PBS), Dimethyl sulfoxide (DMSO), L-glutamine, Sodium bicarbonate, Penicillin (100 U/ml), Streptomycin (100 µg/ml), Amphotericin B (2.5 µg/ml).

Collection of Raw Material

Fresh Gliricidia sepium leaves were identified and then collected in October from the local areas of Kottayam, Kerala. The leaves were shade-dried for five weeks until crispy dry, ground into fine powder using a mechanical grinder, and stored in airtight glass containers for subsequent extraction and in-vitro analysis.

Preparation of Extract

The powdered leaves were extracted by simple maceration using 95% ethanol (1:6 ratio), where 66.66 g of powder was soaked in 400 mL of ethanol. The mixture was shaken continuously for 30 min and macerated for seven days with intermittent shaking. On day seven, it was double-filtered using Whatman filter paper to separate the liquid extract from the marc. The filtrate was then evaporated at 40 °C to remove ethanol. The crude ethanolic extract was stored below 4 °C for subsequent in-vitro studies^[34].

The percentage yield was calculated for the Gliricidia sepium leaf extract based on the crude material used, using the formula:

Percentage Yield (%) = Weight of Plant Material Taken (g) × 100

  Weight of Plant Extract (g)?

In-Vitro Neuroprotective Activity Determination by MTT Assay

Cell Culture

The cell line SHSY-5Y (Neuroblastoma cells) was obtained from the National Centre for Cell Sciences (NCCS) in Pune, India, and was kept in Dulbecco’s Modified Eagle's medium (DMEM) (Sigma Aldrich, USA). The cell line was cultured using DMEM enriched with 10% FBS, L-glutamine, sodium bicarbonate, and an antibiotic solution that included: Penicillin (100 U/ml), Streptomycin (100 µg/ml), and Amphotericin B (2.5 µg/ml). Cell lines were maintained at 37ºC in a 5% CO2 humidified incubator (NBS Eppendorf, Germany)^[35].

Cell Seeding and Compound Stock Preparation

Confluent monolayers of cells that were two days old were trypsinised, and then the cells were resuspended in 10% growth medium. 100 µl suspension of the cell (5x10³ cells/well) was placed in a 96-well tissue culture plate and incubated at 37ºC in a humidified incubator with 5% CO₂. Then, 1mg of the sample was measured and fully dissolved in 1mL DMEM utilizing a cyclomixer. The extract solution was passed through a 0.22 µm Millipore syringe filter to ensure it remained sterile.

Morphological analysis for Neuroprotective activity

Once the cells achieved adequate growth, Beta Amyloid_45-55 (10µM) was introduced to provoke toxicity and was then incubated for one hour^[36]. The 96-well plate contained wells for the different concentrations of sample, control and beta amyloid groups. Following the incubation period, samples of Gliricidia sepium (100, 50, 25, 12.5, and 6.25 µg/mL in DMEM) were applied in triplicate across the respective wells and incubated for 24 hours at 37 °C with 5% CO?. Post-treatment, cells were examined under an inverted phase-contrast microscope (Olympus CKX41 with Optika Pro5 CCD camera), and images were captured for analysis.

Cell viability assay by MTT method

15mg of MTT (Sigma, M-5655) was dissolved in 3 ml PBS, and then sterilized using filter sterilization. Following the 24-hour incubation post-treatment, the contents of the wells were discarded, and 30µl of reconstituted MTT solution was introduced into the wells. The plate was gently agitated and then incubated for 4 hours at 37°C in a humidified 5% CO2 incubator. After the incubation, the supernatant was discarded, and 100µl DMSO was introduced; the wells were then gently shaken to dissolve the formazan crystals produced by the viable cells. The absorbance measurements were taken with a microplate reader at a wavelength of 540 nm^[37]. The percentage cell viability was calculated by the formula:

Percentage cell viability (%) = Mean optical density of sample × 100

        Mean optical density of control

Statistical Analysis

The values obtained after the experiments were expressed as mean ± Standard error mean, where n = 3. The results were analyzed by one-way analysis of variance (ANOVA) followed by Dunnett’s post hoc multiple comparison test. The difference between groups was considered significant at P<0.05*, P<0.01**, P<0.001***, P<0.0001****, and non-significant (NS) at P>0.05 when compared to the β-amyloid group. The statistical analysis was carried out using SPSS software Version 22.

RESULTS

Preparation of Extract and Calculation of the Percentage Yield

The Gliricidia sepium leaves were extracted by the technique of simple maceration using 95% ethanol as solvent. The final percentage yield of the plant extract was found to be 10.2%w/w.

Neuroprotective Activity Studies

The study was carried out to evaluate the in-vitro neuroprotective activity of the ethanolic extract of Gliricidia sepium leaves in the SH-SY5Y human neuroblastoma cell line using β-amyloid _45-55 to induce neurotoxicity. The Aβ-induced cells were treated with five different concentrations of the plant extract (6.25, 12.5, 25, 50, and 100 µg/ml) to assess its neuroprotective effect. The control and β-amyloid wells were also maintained for comparative studies. Cell viability was measured using the MTT colorimetric assay, and microscopical examination was conducted to study the changes in cell morphology. Statistical analysis was conducted using One-way ANOVA followed by Dunnett’s post hoc test.

Effect of Gliricidia sepium Leaf Extract on Cell Morphology

The morphological changes in SH-SY5Y cell lines following Aβ exposure and treatment with different concentrations of the ethanolic extract of Gliricidia sepium leaves were observed through microscopical examination. Control cells displayed normal neuronal morphology with intact and elongated structure with strong adherence to the cell surface, whereas the Aβ group showed shrinkage of cells, dissolution of the cell content, reduced cell density and loss of adherence, exhibiting severe cytotoxicity. Treatment with the plant extract showed a dose-dependent advancement in cell morphology. Partial protection was observed in a lower concentration (12.5 µg/ml). Reduced distorted features of cells were observed in intermediate concentrations (25 and 50 µg/ml). Advanced neuroprotective activity with maximum protection was observed for 100 µg/ml, where the cell morphology was almost similar to that of the control group. Fig. No. 2 illustrates the microscopic images showing the effect of various concentrations of the ethanolic extract of Gliricidia sepium leaves on β-amyloid–induced cytotoxicity in SH-SY5Y cells.

Fig. No. 2: Microscopic images showing the effect of ethanolic extract of Gliricidia sepium leaves on β-amyloid–induced cytotoxicity in SH-SY5Y cells. (a) Control cells showing normal morphology; (b) Aβ-treated cells showing neuronal toxicity; (c-g) GS-treated cells at different concentrations ranging 6.25,12.5,25,50,100 µg/ml showing dose-dependent restoration in cell morphology. The red arrow indicates changes in the morphology of the cell. The green arrow indicates a normal, intact cell.

Effect of Gliricidia sepium Leaf Extract on Cell Viability by MTT Assay

Cell viability of control, β-Amyloid and extracts at various concentrations was assessed using the MTT assay by estimating absorbance at 540nm. The neuroprotective activity of Gliricidia sepium leaf extract is shown in Table 1.

Table 1: Neuroprotective activity of Gliricidia sepium leaf extract

At concentrations 12.5, 25, 50 and 100 µg/ml, Gliricidia sepium extract showed a highly significant (P value < 0.0001****) increase in cell viability to 58.79 ± 1.1329, 63.92 ± 1.3244, 68.02 ± 0.5071 and 71.93 ± 1.2090 %, respectively, when compared to the β-amyloid group, with a cell viability of 49.19 ± 0.2194 %. The concentration 6.25 µg/ml showed a non-significant increase in cell viability (52.57 ± 0.0740 %) with the P value > 0.05. Fig. No. 3 shows the graph obtained after the statistical analysis of the data attained from the experiment.

Fig. No. 3: Effect of ethanolic extract of Gliricidia sepium leaves on percentage cell viability in β-amyloid–treated SH-SY5Y cells by MTT assay. All experiments were performed in triplicate, and the data are expressed as mean ± SEM (n = 3). Statistical analysis was carried out using one-way ANOVA followed by Dunnett’s post hoc test. **** p< 0.0001 compared to β-amyloid–treated group; NS: non-significant (p > 0.05) compared to β-amyloid–treated group.