Evaluation of the Antioxidant and Neuroprotective Properties of Yellow Leaf Extract of Terminalia Catappa In Sleep-Deprived Rats

Sleep is a vital physiological process crucial for maintaining both physical and mental health. Chronic sleep deprivation has been increasingly associated with a range of adverse outcomes, including cognitive decline, mood disorders, and the progression of neurodegenerative diseases (Walker, 2019). As the global prevalence of sleep-related disorders continues to rise, there is growing interest in identifying natural therapeutic agents with neuroprotective properties that can counteract the detrimental effects of inadequate sleep. Herbal remedies have gained attention as promising alternatives to conventional pharmacological treatments for sleep disorders, due to their relatively low risk of dependency and side effects (Asnis et al., 2016). In this context, Terminalia catappa, commonly known as Indian or tropical almond, has emerged as a plant of significant medicinal value. Traditionally used in various cultures, particularly in Asia and the Pacific Islands, the leaves of T. catappa are known to contain a variety of bioactive compounds, including flavonoids, tannins, and saponins, which exhibit antioxidant, anti-inflammatory, and neuroprotective effects (Akinmoladun et al., 2017). Although the therapeutic properties of T. catappa have been explored in treating skin conditions, liver diseases, and metabolic syndromes, its potential for mitigating the neurological effects of chronic stress and sleep deprivation remains relatively underexplored (Chandrasekhar et al., 2017; Zhu et al., 2018). The neuroprotective potential of plant-derived extracts has attracted significant scientific interest in recent years as a complementary approach to managing neurocognitive impairments and neuro inflammation. This study, therefore, seeks to evaluate the antioxidant and neuroprotective properties of yellow leaf extract of Terminalia catappa in a sleep-deprived rat model. By examining its ability to modulate oxidative stress and neurochemical alterations associated with sleep deprivation, this research aims to contribute to the growing body of evidence supporting the use of phytomedicines in the management of sleep-related neurodegenerative disorders.

MATERIALS AND METHODS

Sample Collection and Authentication

Fresh yellow leaves of Terminalia catappa were collected from a mature tree located at Aderibigbe House, Oke Ado Akintola Street, Ogbomoso, Oyo State, Nigeria. Botanical authentication was conducted at the Herbarium Unit, Department of Pure and Applied Biology, Ladoke Akintola University of Technology, Ogbomoso and a voucher specimen was deposited under the accession number LHO 809.

Preparation and Extraction of Plant Material

The yellow leaves of T. catappa collected were thoroughly washed, air-dried at ambient temperature, and pulverized into a fine powder. A 500 g portion of the powdered sample underwent cold maceration in 2.5 L of ethanol for comprehensive phytochemical extraction. The resultant extract was filtered, concentrated under reduced pressure, using a rotary evaporator, and subsequently freeze-dried to obtain the crude ethanol extract.

Plate 1: Fruits, leaves and flower of Terminalia catappa

Determination of Antioxidant Activity

(a). DPPH Radical Scavenging Assay

The antioxidant capacity of the T. catappa yellow leaf extract was evaluated using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay as described by Adeyemo et al. (2018).  0.1 mM DPPH solution in methanol was reacted with various concentrations of the extract (100–500 µg/mL). Each reaction mixture contained 1000 µL of sample and 500 µL of DPPH reagent, incubated in the dark at room temperature for 30 minutes. Absorbance was measured at 518 nm against a methanol blank. Ascorbic acid was used as the reference standard. Percentage inhibition was calculated using:

Where A c = Absorbance of Control and A s = Absorbance of sample.

% Inhibition=Ac- AsAc× 100

(b). Ferric Reducing Antioxidant Power (FRAP) Assay.

The FRAP activity was assessed following Benzie and Strain (1996). Varying concentrations of the extract (100–500 µg/mL) were mixed with 1000 µL of FRAP working reagent and incubated for 6 minutes at room temperature. Absorbance was recorded at 593 nm. A standard curve was generated using ascorbic acid and ferrous sulfate solutions of equivalent concentrations.

FRAP µMvalue =Abs test sampleAbs standard × [FRAP]std (µM)

(c). Hydroxyl Radical Scavenging Assay.

Hydroxyl radical scavenging activity was determined using the phenanthroline-based method (Gulcin et al., 2009). The assay mixture (2.5 mL total volume) contained 1,10-phenanthroline, sodium phosphate buffer, Fe₂ SO?, H?O?, and varying extract concentrations (100–500 µg/mL). After 30-minute incubation at room temperature, absorbance was measured at 536 nm. Ascorbic acid served as the standard. Percentage scavenging was calculated as:

Where AbCtrl= Absorbance of the Control, AbSample= Absorbance of the sample or Standard

Scavenging Activity (%) =    AbCtrl - AbSampleAbs Ctrl × 100

(d). Total Antioxidant Capacity (TAC)

TAC was determined using the phosphomolybdenum method (Baydar et al., 2007). Equal volumes of 0.6 M sulfuric acid, 28 mM sodium phosphate, and 4 Mm ammonium molybdate were mixed to form the reagent. A 100 µL aliquot of extract (100–500 µg/mL) was added to 1000 µL of the reagent and incubated at 95°C for 60 minutes. Absorbance was read at 695 nm after cooling. Antioxidant activity was extrapolated from a standard curve prepared using ascorbic or gallic acid and expressed as mg equivalent per g extract.

Evaluation of Neuroprotective Properties of T. catappa

A. Experimental Animals and Grouping

Female Wistar rats (150–200 g) were procured from the Physiology Department, Ladoke Akintola University of Technology. The rats were kept under standard laboratory conditions (12 h light/dark cycle, ambient temperature and humidity, with ad libitum food and water) and were acclimatized for two weeks. Experimental protocols were approved by the Institutional Animal Ethics Committee. Rats were randomly divided into five groups (n = 7 per group):

Group I: Control (non-sleep deprived)

Group II: Sleep-deprived only (SD)

Group III: Sleep-deprived + Vitamin C (200 mg/kg)

Group IV: Sleep-deprived + extract of T. catappa (100 mg/kg) (D100)

Group V: Sleep-deprived + extract of T. catappa (200 mg/kg) (D200)

Extracts and control solutions were administered orally once daily.

B. Induction of Sleep Deprivation

Sleep deprivation was induced for 12 hours daily (7 a.m.–7 p.m.) over two weeks using the Modified Multiple Platform Method (MMPM). Rats were placed in water tanks (120 × 60 × 45 cm) containing small platforms (6 cm diameter) with water maintained 1 cm below the platform surface. Rats fell into the water upon attempting to enter deep sleep, thus preventing sustained sleep.

C. Post-Deprivation Handling and Tissue Collection

Following the deprivation period, animals were allowed 12 hours of recovery before euthanasia. brain tissues were excised, homogenized, and analyzed for key biochemical markers of neurodegeneration and oxidative stress.

D. Neuro biochemical Assays

(i). Acetylcholinesterase (AChE) Activity

Measured using Ellman method, which detects thiocholine from acetylthiocholine hydrolysis. Thiocholine reacts with DTNB to form a yellow chromophore (412 nm). Enzyme activity was expressed as µmol substrate hydrolyzed per minute per mg protein.

(ii). Glutamate Dehydrogenase (GDH) Activity

Quantified via the reduction of NAD? to NADH during glutamate deamination (Lee & Lardy, 1965). Absorbance was recorded at 340 nm; enzyme activity calculated using NADH’s molar extinction coefficient (6.22 mM?¹ cm?¹).

(iii). Serotonin Quantification

Performed using HPLC with electrochemical detection (Jacobson et al., 1996). Brain homogenates in Perchloric acid were centrifuged and filtered. Serotonin was separated via reverse-phase column and quantified by comparison with standards.

(iv). Glutathione (GSH) Concentration

Measured by reaction with DTNB forming 5-thio-2-nitrobenzoate, detectable at 412nm (Beutler et al., 1963). The values were compared to a standard GSH calibration curve.