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

Akintorinwa O., Ayoola P. B., Kehinde B. D., Akintola A. O.,

doi:10.5281/zenodo.16760660

Research Paper | Open Access
Volume 02 | Issue 08 | Article Id IJSRT/250307069

Evaluation of the Antioxidant and Neuroprotective Properties of Yellow Leaf Extract of Terminalia Catappa In Sleep-Deprived Rats
Akintorinwa O.* Ayoola P. B. Kehinde B. D. Akintola A. O.
Department of Science Laboratory Technology, Ladoke Akintola University of Technology, Ogbomoso, P.M.B 4000, Ogbomoso, Oyo State, Nigeria

Abstract

Sleep deprivation is increasingly recognized as a public health issue linked to cognitive dysfunction and neurodegeneration due to oxidative stress. This study explores the neuroprotective potential of yellow leaf extract of Terminalia catappa (T. catappa) in a rat model of sleep deprivation. Samples were obtained from oke ado akintola area in Ogbomoso, Southwestern, Nigeria. It was washed, air dried and pulverized. Extraction was done using ethanol, the extract was concentrated using a rotary evaporator. 35 female wistar rats were used, which were acclimatized for 2 weeks and different dosages of extracts were administered for 2 weeks. The rats were divided into five groups: Group I (control), Group II (sleep-deprived untreated), Group III (sleep deprived + Vitamin C), Group IV (sleep-deprived + 100mg/kg of T. catappa extract) and Group V (sleep-deprived + 200mg/kg of T. catappa extract). Sleep was induced in rats using Modified Multi-Platform Method (MMPM) and was sacrificed using cervical dislocation; the brain tissue was collected for assay. Biochemical assays revealed that T. catappa caused significant biochemical changes. It decreases level of Acetylcholinesterase (AchE), Glutamate dehydrogenase (GDH), Malondialdehyde (MDA), and Nitric oxide (NO). Concurrently, it increases levels of Serotonin, Glutathione (GSH), and Superoxide dismutase (SOD). These findings suggest T. catappa yellow leaf extract as a potent neuroprotective agent with therapeutic promise in oxidative stress-related neural conditions.

Keywords

Terminalia catappa, sleep deprivation, oxidative stress, antioxidants, neuroprotection, Wistar rats

Introduction

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.

Plate 2: Modified Multi-Platform Method (MMPM),

(v). Superoxide Dismutase (SOD) Activity

Assessed based on the enzyme’s ability to inhibit the auto-oxidation of epinephrine at pH 10.2 (Misra & Fridovich, 1972). Absorbance was measured at 480 nm. One unit of SOD was defined as the amount causing 50% inhibition of epinephrine oxidation.

(vi). Nitric Oxide (NO) Concentration

Determined via the Griess reaction, which detects nitrite as a stable end-product of NO metabolism (Grisham et al., 1996). The pink azo compound formed was measured at 540 nm and quantified using sodium nitrite standards.

(vii). Malondialdehyde (MDA) Level

Assessed using the TBARS assay (Ohkawa et al., 1979). MDA reacts with thiobarbituric acid under high heat to form a colored complex detectable at 532 nm. Concentrations were derived from a standard curve of tetraethoxypropane.

RESULTS AND DISCUSSION

Antioxidant Activity of Yellow Leaf Extract of Terminalia catappa

The antioxidant capacity of Terminalia catappa yellow leaf extract was evaluated using hydroxyl radical scavenging, DPPH radical scavenging, and ferric reducing antioxidant power (FRAP) assays, all of which revealed notable activity in a concentration-dependent manner. In the hydroxyl radical scavenging assay, the extract demonstrated moderate activity with a maximal inhibition of 56.87% at 250 µg/mL and an IC?? of 209.81 µg/mL. These results, while indicating appreciable radical neutralization potential, suggest moderate potency compared to more commonly studied antioxidants such as Azadirachta indica (IC?? = 180 µg/mL) (Alzohairy, 2016) and Moringa oleifera (Verma et al., 2009). The decreasing absorbance values with increasing extract concentration reflect effective scavenging of hydroxyl radicals. The FRAP assay further confirmed the extract’s robust antioxidant capacity, with a reducing power of 217.56 ± 1.01 mM Fe²? equivalents/g dry weight. This value exceeds those reported for M. oleifera and green tea (Carloni et al., 2013), approaching the antioxidant efficacy of ascorbic acid (Benzie & Strain, 1996). The high and reproducible FRAP activity suggests the presence of potent electron- donating compounds and underlines the extract' s potential for nutraceutical or functional food applications. The DPPH radical scavenging assay also demonstrated strong activity, with inhibition ranging from 48.05% to 57.63% across the 50–250 µg/mL concentration range, and an IC?? of 100.25 µg/mL. This value is notably lower than that of Ocimum sanctum (Prakash & Gupta, 2005) and comparable to Terminalia chebula (Bag et al., 2013), suggesting significant free radical neutralization via hydrogen-donating mechanisms. The superior DPPH scavenging relative to hydroxyl radical scavenging suggests specificity toward nitrogen-centered radicals, consistent with the behavior of polyphenol-rich extracts (Prior et al., 2005). Collectively, the results highlight the yellow leaf extract of T. catappa as a promising source of natural antioxidants with broad-spectrum radical scavenging and reducing properties. These findings support its potential use in mitigating oxidative stress- related pathologies, warranting further bioactivity-guided phytochemical characterization.

Table 1: Hydroxyl Radical Scavenging activity of Leaf Extract of T. catappa

S/N	Concentration (µg/mL)	% Radical scavenging	IC 50 (µg/mL)
1.	50	29.5669	209.806
2.	100	32.9567
3.	150	40.8663
4.	200	48.0226
5.	250	56.8738

Table 2: Ferric Reducing Antioxidant Power (FRAP) of Leaf of T. catappa

	FRAP (mM Fe 2+ /g)
T. catappa Extract	217.56 ± 1.01
Ascorbic Acid*	250.18 ± 0.85

Table 3: DPPH Radical Scavenging Activity of Leaf Extract of T. catappa

S/N	Concentration (µg/mL)	% Radical scavenging	IC 50 (µg/mL)
1.	50	48.0465	100.2545
2.	100	49.8605
3.	150	51.814
4.	200	54.51165
5.	250	57.6279

Modulation of Acetylcholinesterase (AChE) Activity

This study evaluated the neuroprotective potential of Terminalia catappa leaf extract on AChE activity in sleep-deprived rats. Sleep deprivation significantly increased AChE activity in the hippocampus (Group II) compared to the control group (Group I), indicating cholinergic disruption associated with cognitive impairment. Treatment with T. catappa at 100 mg/kg and 200 mg/kg (Groups IV and V) significantly reduced AChE activity in a dose-dependent manner (p < 0.05), suggesting restoration of cholinergic function, likely through antioxidant mechanisms. Similarly, Vitamin C (Group III) also reduced AChE activity, consistent with its known antioxidant role. Given Vitamin C’s well-established antioxidant properties, the similar trend observed with T. catappa highlights its therapeutic potential in preserving acetylcholine availability and improving cognitive resilience to sleep loss.

Figure 1: Effect of T. catappa Leaf Extract on Acetylcholinesterase (AchE) Activity in Sleep Deprived Rats