INTRODUCTION:

Neurodegenerative diseases like Parkinson's disease (PD), Alzheimer's disease (AD), and Huntington's disease (HD) pose a serious threat to public health, globally. Progressive neuronal death, synaptic malfunction, and cognitive impairment are the hallmarks of these illnesses.¹

Therefore, one therapeutic approach to reduce or stop the progression of neurodegenerative illnesses is to target neuroinflammation. A promising strategy for neuroprotection is the investigation of natural compounds having anti-inflammatory and antioxidant qualities.

According to recent research, plant extracts such as Bacopa monnieri, Withania somnifera, and Curcuma longa enhance cognitive function and shield neurons from inflammation and oxidative damage.² From the Apocynaceae family, Tabernaemontana divaricata is a medicinal plant which contains indole alkaloids, flavonoids, terpenoids, and phenolic chemicals in abundance. Its anti-inflammatory, antioxidant, neuroprotective, analgesic, and wound-healing properties have been verified by several experimental and ethnomedical investigations.³ T. divaricata exhibits neuroprotective effect through a number of mechanisms which may include:

· Antioxidant activity: Through alkaloids and flavonoids that scavenge free radicals, stop lipid peroxidation, and boost antioxidant enzymes like SOD, CAT, and GSH, T. divaricata demonstrates strong antioxidant activity. This process lessens oxidative stress-related neuronal damage while maintaining the integrity of the neuronal membrane and mitochondrial function ⁴.

· Anti-inflammatory effect: T. divaricata contains bioactive substances that inhibit pro-inflammatory cytokines like TNF-α, IL-1β, and IL-6 and reduce NF-κB activation. This prevents neuronal apoptosis and shields hippocampus neurons from LPS-induced injury by lowering microglial activity and neuroinflammation ⁵.

· Cholinergic modulation: T. divaricata improves cognitive function by raising acetylcholine levels by blocking acetylcholinesterase. Voacangine and coronaridine are examples of indole alkaloids that enhance neuronal transmission and memory processing by modulating muscarinic and nicotinic receptors ⁶.

· Neurotrophic support: T. divaricata's antioxidant activity keeps BDNF levels stable, protecting synaptic plasticity and neuronal shape. Under oxidative or inflammatory stress, this neurotrophic impact promotes learning and memory by supporting neuronal survival and regeneration in the hippocampus ⁷. When taken as a whole, these mechanisms imply that T. divaricata may provide complex neuroprotection against LPS-induced neuroinflammatory and cognitive deficits.

T. divaricata has shown a variety of pharmacological properties, but little research has been done on how it affects neuroinflammation-induced cognitive impairment. An excellent platform for assessing T. divaricata's neuroprotective and cognitive-enhancing capabilities is the LPS-induced model of neuroinflammation. Examining the plant's ability to prevent LPS-induced spatial memory loss may help determine how it may be used therapeutically to treat neurodegenerative diseases like Alzheimer's.

MATERIALS AND METHOD:

During the post-harvest season (November–December), when the foliage was mature and phytochemically stable, Tabernaemontana divaricata leaves were gathered from the local area of Chhattisgarh, specifically from the Bilaspur District, India. A botanist from Guru Ghasidas University's Department of Botany in Bilaspur, Chhattisgarh, India, taxonomically verified the plant material using both macroscopic and microscopic diagnostic traits. A voucher specimen (Reference No. CP/PHAR/2025/01) was prepared and deposited in the departmental herbarium for future verification and research use.

500mL of petroleum ether (60–80°C) was used to extract 100g of dried and powdered Tabernaemontana divaricata leaves for 4–6 hours. The leaves were then submitted to Soxhlet extraction using 70% ethanol (1 L) for 48 hours until all phytoconstituents had been exhausted.⁸ Yield was assessed. The ethanolic extract underwent a preliminary phytochemical screening.

Approval for Animal Experimentation and Acclimatization:

In compliance with CPCSEA rules, the experimental protocol was authorized by the Institutional Animal Ethics Committee (IAEC) of the School of Pharmacy, Chouksey Engineering College, Bilaspur, Chhattisgarh (Approval No. SOP/IAEC/2024/11/14). A CPCSEA-registered animal facility provided healthy adult Wistar rats weighing between 180 and 220grams.⁹ Clean polypropylene cages with a 12-hour light/dark cycle, a temperature of 22±2°C, and a relative humidity of 55–65% were employed to keep the animals.¹⁰

Acute Oral Toxicity Study:

Using both male and female Wistar rats (150–180g), an acute oral toxicity investigation of the Tabernaemontana divaricata hydroalcoholic extract and its alkaloid component was conducted in accordance with OECD Guideline 423.

Neuroprotective Assessment Against LPS-Induced Neuroinflammation and Spatial Memory Impairment in Rats:

In this work, a single intraperitoneal injection of LPS at a dosage of 0.05mg/kg caused neuroinflammation. Five experimental groups, each consisting of six animals, were randomly selected from a total of twenty-five healthy Wistar rats^11-12.

Experimental Groups (n = 5 each)

Group I – Normal Control: Received normal saline orally for 14 days via gavage.

Group II – LPS Control: Received LPS (0.05mg/kg, i.p.) without any plant extract treatment.

Group III – LPS + T. divaricata (200mg/kg): Received T. divaricata leaf extract orally (200mg/kg/day) for 14 days along with LPS (0.05mg/kg, i.p.).

Group IV – LPS + T. divaricata (400 mg/kg): Received T. divaricata leaf extract orally (400 mg/kg/day) for 14 days along with LPS (0.05mg/kg, i.p.).

Group V – LPS + Galantamine: Received Galantamine (2.5mg/kg/day, p.o.) as standard drug along with LPS (0.05mg/kg, i.p.).

Behavioral Evaluation:

The Y-Maze Test and the Novel Object Recognition Test (NORT) were used for behavioral evaluation.

Pathological Evaluation:

Using commercially available rat-specific kits, the levels of pro-inflammatory cytokines (including IL-6) in brain homogenates were assessed by ELISA. The Thiobarbituric Acid Reactive Substances (TBARS) assay was used to measure MDA levels in order to evaluate oxidative damage resulting from LPS-induced neuroinflammation. The traditional Ellman method was used to test AChE activity in brain tissue homogenates in order to assess cholinergic dysfunction. Additionally, a histopathological study was carried out.

RESULTS AND DISCUSSION:

1. Extractive Value:

It was discovered that Tabernaemontana divaricata leaves had an extraction value of 18.6% w/w in 70% ethanol. This stated that the leaf powder included a considerable amount of ethanol-soluble phytochemicals and demonstrated how effective the hydroalcoholic solvent system was in dissolving and extracting the phytoconstituents found in the plant material.

Table no. 5.1 Extractive Value of Tabernaemontana divaricata leaves (70% ethanol)

Solvent	Extractive Value (% w/w)
Ethanol	18.6%

Fig. no. 5.1 Extraction of Tabernaemontana divaricata leaves

2. Phytochemical Screening of Active Phyto-compounds:

Several significant groups of secondary metabolites were found in the hydroalcoholic extract of Tabernaemontana divaricata leaves, according to an initial phytochemical screening. Overall, the phytochemical profile indicated that the extract was abundant in bioactive metabolites, including terpenoids, alkaloids, flavonoids, and saponins, which may all work together to support the extract's pharmacological effects.

Table no. 5.2 Phytochemical Constituents in Hydroalcoholic Extract of T. divaricata Leaves

Phytochemicals	Test Performed	Observations	Ethanolic Extract
Alkaloids	Dragendorff’s test	Orange Red Precipitate	+++ (abundant)
Flavonoids	Shinoda test	Pink–red coloration	++ (moderate)
Saponins	Foam test	Stable persistent froth	++
Tannins	Ferric chloride test	Blue-black coloration	± (trace)
Terpenoids	Salkowski test	Reddish-brown interface	++

Where: (+ = present, ± = trace, - = absent)

3. Fractionation of Active Phyto-compounds: Alkaloid Fractionation:

The alkaloid fraction obtained showed a yield of 3.8% w/w. This yield indicated that the plant possessed a measurable and appreciable quantity of alkaloidal constituents capable of being selectively extracted through acid–base fractionation. This provided a valuable basis for subsequent phytochemical characterization and bioactivity studies.

Table no. 5.3 Yield of Alkaloid Fraction from Ethanolic Extract of T. divaricata Leaves

Fraction Type	Yield (%w/w)
Alkaloid Fraction	3.8%

Fig. no. 5.2 Alkaloid Fraction from Ethanolic Extract of T. divaricata Leaves

4. Acute Oral Toxicity Study:

The hydroalcoholic extract and alkaloid fraction of T. divaricata leaves are non-toxic at a high dose of 2000 mg/kg, according to the acute oral toxicity study. Furthermore, gross pathological analysis verified that the extracts do not negatively impact major organs, which is consistent with earlier findings about T. divaricata's low toxicity profile. These results lay the groundwork for employing these extracts in preclinical research without running the risk of acute toxicity.

Table no. 5.4 Acute Oral Toxicity Study of Tabernaemontana divaricata Extract

Parameter	Control Group	Hydroalcoholic Extract (2000mg/kg)	Alkaloid Fraction (2000mg/kg)	Result and Discussion
Mortality (14 days)	0/6	0/6	0/6	No mortality observed; LD50 estimated > 2000 mg/kg indicating safety
Clinical Signs (0–4 h)	Normal	No signs of toxicity; normal behaviour	No signs of toxicity; normal behaviour	Extracts showed no acute behavioural abnormalities
Clinical Signs (24 h – 14 days)	Normal	No changes in posture, gait, respiration, salivation, tremors, or convulsions	No alterations observed	Both samples were well tolerated throughout study duration
Body Weight (g) Day 0	158.3 ± 4.2	160.1 ± 3.8	159.6 ± 4.1	No significant difference in initial weight
Body Weight (g) Day 7	165.4 ± 4.5	167.2 ± 3.9	166.8 ± 4.0	Normal weight gain pattern observed
Body Weight (g) Day 14	172.8 ± 5.1	174.6 ± 4.7	175.1 ± 4.3	Weight gain similar to control, indicating no metabolic disturbances
Food Intake	Normal	Normal	Normal	No appetite suppression or toxicity related survival
Survival	100%	100%	100%	All animals survived → confirms absence of acute toxicity
Gross Pathology	Normal	No abnormalities in liver, kidney, heart. Lungs, stomach and spleen	No abnormalities	No organ-specific toxicity detected, confirming extract safety
Overall Outcome	-	Safe at 2000 mg/kg	Safe at 2000 mg/kg	Extract and alkaloid fraction are non toxic and suitable for further neuroprotective studies

Table no. 5.5. Y-Maze Test – Spontaneous Alternation (%)

Group	Treatment	R1	R2	R3	R4	R5	Alteration % (Mean ± SEM)
I	Normal Control	72.5	73.6	71.9	74.2	72.8	73.0 ± 0.38
II	LPS Control	41.3	42.6	43.0	39.9	41.5	41.6 ± 0.54 a****
III	LPS + Galantamine	63.1	62.5	64.4	63.8	62.8	63.3 ± 0.36 a** b**
IV	LPS + Extract 200 mg/kg	55.7	54.1	55.2	56.1	54.8	55.1 ± 0.32 a** b**
V	LPS + Extract 400 mg/kg	59.4	58.1	59.8	60.2	58.7	59.2 ± 0.39 a** b**

Values = Mean ± SEM, n=5. Where a = when compared with Normal Control and b = when compared with LPS (Toxic Control), R = Replicant Animal, **** = p < 0.0001.

Table no. 5.6. Novel Object Recognition Test (%)

Group	Treatment	R1	R2	R3	R4	R5	Locomotor Activity (Mean ± SEM)
I	Normal Control	0.62	0.65	0.61	0.63	0.64	0.63 ± 0.01
II	LPS Control	0.22	0.25	0.21	0.23	0.24	0.23 ± 0.01 a****
III	LPS + Galantamine	0.49	0.47	0.48	0.50	0.49	0.48 ± 0.01 a** b**
IV	LPS + Extract 200 mg/kg	0.38	0.36	0.37	0.39	0.38	0.38 ± 0.01 a** b**
V	LPS + Extract 400 mg/kg	0.44	0.45	0.43	0.46	0.45	0.44 ± 0.01 a** b**

Values = Mean ± SEM, n=5. Where a = when compared with Normal Control and b = when compared with LPS (Toxic Control), R = Replicant Animal, **** = p < 0.0001.

Table no. 5.7. Novel Object Recognition Test (%)

Group	Treatment	R1	R2	R3	R4	R5	IL6 (Mean ± SEM)
I	Normal Control	18.4	19.1	17.9	18.8	18.1	18.4 ± 0.20
II	LPS Control	52.8	54.2	53.6	55.1	52.9	53.7 ± 0.39 a****
III	LPS + Galantamine	31.4	30.8	30.5	32.0	31.1	31.2 ± 0.25 a** b**
IV	LPS + Extract 200 mg/kg	38.2	37.8	39.1	38.6	37.9	38.3 ± 0.23 a** b**
V	LPS + Extract 400 mg/kg	34.7	33.8	34.2	35.1	34.3	34.4 ± 0.18 a** b**

Values = Mean ± SEM, n=5. Where a = when compared with Normal Control and b = when compared with LPS (Toxic Control), R = Replicant Animal, **** = p < 0.0001

Table no. 5.8. Novel Object Recognition Test (%)

Group	Treatment	R1	R2	R3	R4	R5	MDA (Mean ± SEM)
I	Normal Control	2.8	2.9	3.0	2.7	2.9	2.86 ± 0.05
II	LPS Control	6.1	6.4	6.3	6.0	6.5	6.26 ± 0.08 a****
III	LPS + Galantamine	4.1	4.3	4.0	4.2	4.1	4.14 ± 0.05 a** b**
IV	LPS + Extract 200 mg/kg	5.0	4.9	5.1	5.2	5.0	5.04 ± 0.05 a** b**
V	LPS + Extract 400 mg/kg	4.5	4.6	4.4	4.3	4.5	4.46 ± 0.04 a** b**

Values = Mean ± SEM, n=5. Where a = when compared with Normal Control and b = when compared with LPS (Toxic Control), R = Replicant Animal, **** = p < 0.0001

Table no. 5.9. Novel Object Recognition Test (%)

Group	Treatment	R1	R2	R3	R4	R5	Locomotor Activity (Mean ± SEM)
I	Normal Control	0.42	0.44	0.43	0.41	0.42	0.42 ± 0.01
II	LPS Control	0.88	0.85	0.87	0.89	0.86	0.87 ± 0.01 a****
III	LPS + Galantamine	0.52	0.51	0.53	0.54	0.52	0.52 ± 0.01 a* b**
IV	LPS + Extract 200 mg/kg	0.64	0.63	0.65	0.66	0.64	0.64 ± 0.01 a** b*
V	LPS + Extract 400 mg/kg	0.57	0.56	0.58	0.59	0.57	0.57 ± 0.01 a* b**

Values = Mean ± SEM, n=5. Where a = when compared with Normal Control and b = when compared with LPS (Toxic Control), R = Replicant Animal, *** = p < 0.001 and **** = p < 0.0001

Fig, no. 5.7. AChE Activity (µmol/min/mg protein)

5.3 Histopathological Evaluation:

The hippocampus and cortex were examined histopathologically, and the results showed clear morphological differences between the experimental groups.

Improved behavioral results and normalized biochemical indicators are consistent with the preservation of neuronal architecture, indicating that the extract reduces oxidative stress and neuroinflammation brought on by LPS.

Table no. 5.10. Histopathological Evaluation of Brain Sections

Group	Histopathological Observations
Normal Control	Normal neurons; no degeneration or inflammation
LPS Control	Neuronal degeneration, cell loss, and inflammatory infiltration
LPS + Galantamine	Improved neuronal structure; minimal inflammation
LPS + T. divaricata Extract (200 mg/kg)	Partial restoration; moderate reduction in degeneration and inflammation
LPS + T. divaricata Extract (400 mg/kg)	Significant preservation; minimal degeneration and inflammation; dose-dependent protection

Fig. no. 5.8 Representative HandE-stained brain sections of Rat: a. Normal: intact neurons, b. LPS: neuronal necrosis, vacuolation, c. LPS + Galantamine, d. LPS + Extract 200 mg/kg: partial neuronal protection and e. LPS + Extract 400 mg/kg: near normal architecture

CONCLUSION:

The goal of the current study was to assess Tabernaemontana divaricata leaf extract's neuroprotective potential against lipopolysaccharide (LPS)-induced neuroinflammation and spatial memory impairment in rats. In order to simulate the clinical characteristics of neurodegenerative illnesses, LPS administration effectively caused neuroinflammation, resulting in oxidative stress, raised pro-inflammatory cytokine levels, and impairment in spatial memory. Treatment with T. divaricata extract significantly reduced LPS-induced cognitive impairments, demonstrating its protective effect on learning and memory functions, according to behavioral assessment utilizing spatial memory tests.

Overall, the results of this study demonstrate Tabernaemontana divaricata's potential as a natural therapeutic agent for the treatment of neuroinflammation-related cognitive impairment and offer scientific evidence in favor of its traditional usage in neurological illnesses.

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Received on 18.03.2026 Revised on 06.04.2026

Accepted on 20.04.2026 Published on 22.04.2026

Available online from April 24, 2026

Res.J. Pharmacology and Pharmacodynamics.2026;18(2):140-146.

DOI: 10.52711/2321-5836.2026.00019

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